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T4 endonuclease V [endodeoxyribonucease (pyrimidine dimer); deoxyribonuclease (pyrimikine dimer), EC 3.1.25.1] initiates repafr of damaed DNA by hydrolysis of the N-glycosyl bond at the 5' side of a pyrimidine photodimer in double-stranded DNA. To study one of the active sites of T4 endonudease V, systematic site-directed mutagenesis was performed on the synthetic T4 endonuclease V gene, in paraflel with three-dimensional structure analysis by x-ray crystallography. The mutant proteins were evaluated for DNA glycosylase activity using an oligonucleotide duplex (14-mer) containing a single thymidine dimer as a substrate. Replacement of either Glu-23 with glutamine or asparatic acid or Arg-3 with glutami completely abolished DNA glycosylase activity. Mutation of Arg-3 to lysine or of Arg-26 to glu or lysine in a basic amino acid cluster caused serious defects in DNA glycosylase activity, which are reflected in the increases in K. and decreases in kt of DNA glycosylase activity. On the other hand, substitutions of lysine for Arg-22 or of glutamine for Arg-117 or Lys-121 resulted in increases in the Km value. The completely inactive mutant proteins, E23Q and R3Q, in which glutamie was substituted for Glu-23 and Arg-3, respectively, were further investigated by CD spectroscopy for their ability to bind the oligonucleotide substrate. It was found that the E23Q protein retained specific substrate-binding ability, whereas the R3Q protein did not. These results indicate that Glu-23 plays an important role in catalysis of the DNA glycosylase reaction, and that Arg-3 is a crucial residue for substrate binding. In addition, Arg-22, Arg-26, Arg-117, and Lys-121 in the basic amino acid cluster also participate in substrate binding. We conclude that the basic amino acid cluster in T4 endonuclease V is an essential structure for DNA glycosylase activity.Endonuclease V [endo V; endodeoxyribonuclease (pyrimidine dimer); deoxyribonuclease (pyrimidine dimer), EC 3.1.25.1], found in bacteriophage T4, is responsible for excision repair of damaged DNA. The enzyme possesses two activities: a pyrimidine dimer DNA glycosylase (PD glycosylase) and an apyrimidinic/apurinic (AP) endonuclease ( Fig. 1) (1, 2). The gene (T4 denV) encoding T4 endo V was identified in association with the UV resistance of T4 phage and was shown to encode 138 amino acids (Fig. 2) (3). The protein recognizes a pyrimidine photodimer structure in a double-stranded DNA, hydrolyzes the N-glycosyl bond at the 5' side of a pyrimidine dimer, and cleaves the apyrimidinic phosphodiester bond via a (3-elimination reaction. In addition, T4 endo V is capable of binding and scanning nontarget DNA processively (4). The fact that such a small protein possesses two catalytic activities has engendered considerable interest in terms of the relationship between the two activities and the domain structure. As yet, no catalytic mechanism of any PD glycosylase has been elucidated.The aromatic residue-rich region, Trp-Tyr-Lys-Tyr-Tyr (residues 128-132), near the C terminus of ...

Multicellular tumor spheroids (MCTS) are a reliable model of nonvascularized tumor cell aggregates showing a well defined three-dimensional growth pattern and are comparable with small metastatic cancer cell complexes in blood circulation. In the present study we have established a co-culture system of multicellular bladder tumor spheroids with human endothelial cells on extracellular matrix (ECM) in order to investigate morphological and proliferative changes of endothelial and tumor cells within a defined time of cell-cell interaction. The MCTS--endothelial cell--extracellular matrix complex was observed within coculture periods from 1/2 to seven days. Morphological changes (light microscopy, scanning and electron microscopy) indicated that MCTS are not influenced by cocultured endothelial cells. The tumor cells invaded into the ECM after degradation of endothelial cells in the center of the contact zone. Endothelial cells, however, showed degenerative changes as well as a complex reaction in their proliferation activities. We could recognize an initial increase of proliferation of endothelial cells next to the MCTS. Later on, endothelial cells next to invading tumor cells showed changes in morphological polarity. The model system used has the advantage of using human tumor tissue. It distinguishes between basic cellular mechanisms like adherence, migration, DNA synthesis and proliferation in the study of the contact of tumor cells and vascular endothelial cells as an important event in hematogenous tumor spread.

Realistic boundaries of finite heat conductivity for thermoconvection in a

The statistical dynamics of fluctuating Taylor vortex flow (TVF) and Rayleigh-Bénard convection (RBC) in the presence of externally imposed throughflow is studied in the convectively unstable regime in the presence of thermal noise. A relation between thermal fluctuations at the inlet and downstream vortex flow is derived by coarse-graining appropriate microscopic fields. The downstream growth of macroscopic vortices out of molecular fluctuations is investigated and relevant fluctuation spectra are evaluated.