We report on investigations aimed at detecting mutated RAS genes in a variety of preleukemic disorders (1,5,6). Moreover, in all cases investigated thus far, the mutations turned out to be specific for the tumor cells and were not found in normal cells of the respective patient. Activation of RAS genes has been detected in a variety of different neoplasias with variable frequencies. By far, the highest incidence (25-50%) has been reported in acute myelocytic leukemia (AML) (6-8).To gain further information on the biological significance of RAS mutations in leukemogenesis, we investigated the occurrence of RAS gene mutations in AML compared to a broad spectrum of other preleukemic and leukemic disorders involving the myeloid lineage. For this purpose we used, in addition to DNA transfection analyses (tumorigenicity assay), a dot-blot screening procedure based on a combination of in vitro amplification of RAS-specific sequences and hybridization to mutation-specific oligonucleotide probes (9)(10)(11)

A 34-mer oligonucleotide containing a single 7,8-dihydro-8-oxoguanine (8-OxoG) residue was used to study the enzymatic and DNA binding properties of the Fpg protein from E. coli. The highest rates of incision of the 8-OxoG containing strand by the Fpg protein were observed for duplexes where 8-OxoG was opposite C (*G/C) or T (*G/T). In contrast, the rates of incision of duplexes containing 8-OxoG opposite G (*G/G) and A (*G/A) were 5-fold and 200-fold slower. Gel retardation studies showed that the Fpg protein had a strong affinity for duplexes where the 8-OxoG was opposite pyrimidines and less affinity for duplexes where the 8-OxoG was opposite purines. KDapp values were 0.6 nM (*G/C), 1.0 nM (*G/T), 6.0 nM (*G/G) and 16.0 nM (*G/A). The Fpg protein also binds to unmodified (G/C) duplex and a KDapp of 90 nM was measured. The cleavage and binding of the (*G/C) duplex were also studied using bacterial crude lysates. Wild type E. coli crude extract incised the 8-OxoG containing strand and formed a specific retardation complex with the (*G/C) duplex. These two reactions were mediated by the Fpg protein, since they were not observed with a crude extract from a bacterial strain whose fpg gene was inactivated. Furthermore, we have studied the properties of 6 mutant Fpg proteins with Cys-->Gly mutations. The results showed that the 2 Fpg proteins with Cys-->Gly mutations outside the zinc finger sequence cleaved the 8-OxoG containing strand, formed complexes with the (*G/C) duplex and suppressed the mutator phenotype of the fpg-1 mutant. In contrast, the 4 Fpg proteins with Cys-->Gly mutations within the zinc finger motif neither cleave nor bind the (*G/C) duplex, nor do these proteins suppress the fpg-1 mutator phenotype.

A series of poly(R-3-hydroxybutyrate)/poly(-caprolactone)/1,6-hexamethylene diisocyanate-segmented poly(ester-urethanes), having different compositions and different block lengths, were synthesized by one-step solution polymerization. The molecular weight of poly(R-3-hydroxybutyrate)-diol, PHB-diol, hard segments was in the range of 2100 -4400 and poly(-caprolactone)-diol, PCL-diol, soft segments in the range of 1080 -5800. The materials obtained were investigated by using differential scanning calorimetry, wide angle X-ray diffraction and mechanical measurements. All poly(ester-urethanes) investigated were semicrystalline with T m varying within 126 -148°C. DSC results showed that T g are shifted to higher temperature with increasing content of PHB hard segments and decreasing molecular weight of PCL soft segments. This indicates partial compatibility of the two phases. In poly(ester-urethanes) made from PCL soft segments of molecular weight (M n Ն 2200), a PCL crystalline phase, in addition to the PHB crystalline phase, was observed. As for the mechanical tensile properties of poly(ester-urethane) cast films, it was found that the ultimate strength and the elongation at the breakpoint decrease with increasing PHB hard segment content.

A method is described that demonstrates a new technique for rapid and high‐throughput single molecule sequencing. This sequencing technique is based on the successive enzymatic degradation of fluorescently labeled single DNA molecules, and the detection and identification of the released monomer molecules according to their sequential order in a microstructured channel. The detection technique is evolved from confocal fluorescence microscopy, with two different laser sources to excite the individual mononucleotides that are either labeled with tetramethylrhodamine (TMR) or Cyanine5 (Cy5). The handling of DNA which is immobilized on carrier beads, and the detection of the cleaved monomers is performed in optically transparent and biochemically inert microstructures (glass or PMMA) with detection channels of 7 μ × 10 μm. The projected rate of sequencing is ≈100 bases min−1, dependent solely on the rate of the enzymatic DNA cleavage.

The C2 toxin of Clostridium botulinum is a binary bacterial protein toxin, comprising the enzyme component C2I and the separate binding/translocation component C2IIa. C2IIa mediates the transport of C2I into the host cell cytosol. The N-terminal domain of C2I (C2IN) is enzymatically inactive but essential for C2IIa-mediated internalization of C2I. Here, we exploit the C2IIa/C2IN system to generate a recombinant C2IN-streptavidin fusion protein allowing for the delivery of biotinylated molecules into the cytosol of mammalian cells. C2IN-streptavidin overproduced in E. coli was affinity-purified and capable of binding biotinylated proteins in a concentration-dependent manner. Real-time surface plasmon resonance confirmed the biotin-mediated interaction yielding a K(D)-value of approximately 0.75 muM. Internalization of C2IN-streptavidin into the cytosol of epithelial cells and macrophages was demonstrated by immunoblot analysis and confirmed by confocal microscopy. Cell viability studies showed no cytotoxic effects of the novel transporter. Furthermore, Vero cells treated with biotin-fluorescein or biocytin-Alexa488 as model cargo displayed a specific C2IN-streptavidin/C2IIa-dependent uptake, providing proof-of-principle for the functionality of this novel delivery system.