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Repair System for Noncanonical Purines in Escherichia coli. Nicholas E. Burgis, 2003.Exposure of Escherichia coli strains deficient in molybdopterin biosynthesis (moa) to the purine base N-6-hydroxylaminopurine (HAP) is mutagenic and toxic . We show that moa mutants exposed to HAP also exhibit elevated mutagenesis, a hyperrecombination phenotype, and increased SOS induction . The E . coli rdgB gene encodes a protein homologous to a deoxyribonucleotide triphosphate pyrophosphatase from Methanococcus jannaschii that shows a preference for purine base analogs . moa rdgB mutants are extremely sensitive to killing by HAP and exhibit increased mutagenesis, recombination, and SOS induction upon HAP exposure . Disruption of the endonuclease V gene, nfi, rescues the HAP sensitivity displayed by moa and moa rdgB mutants and reduces the level of recombination and SOS induction, but it increases the level of mutagenesis . Our results suggest that endonuclease V incision of DNA containing HAP leads to increased recombination and SOS induction and even cell death . Double-strand break repair mutants display an increase in HAP sensitivity, which can be reversed by an nfi mutation . This suggests that cell killing may result from an increase in double-strand breaks generated when replication forks encounter endonuclease V-nicked DNA . We propose a pathway for the removal of HAP from purine pools, from deoxynucleotide triphosphate pools, and from DNA, and we suggest a general model for excluding purine base analogs from DNA . The system for HAP removal consists of a molybdoenzyme, thought to detoxify HAP, a deoxyribonucleotide triphosphate pyrophosphatase that removes noncanonical deoxyribonucleotide triphosphates from replication precursor pools, and an endonuclease that initiates the removal of HAP from DNA . Novel Polysulfated Galactose-Derivatized Dendrimers as Binding Antagonists of Human Immunodeficiency Virus Type 1 Infection. Richard D. Kensinger, 2004.Evidence indicates that galactosyl ceramide (GalCer) and its 3'-sulfated derivative, sulfatide (SGalCer), may act as alternate coreceptors for human immunodeficiency virus type 1 (HIV-1) in CD4– cells . Glycosphingolipids (GSLs) may also be necessary for fusion of HIV-1 and host cell membranes . Using an enzyme-linked immunosorbent assay to determine which GSL was the best ligand for both recombinant and virus-associated gp120, we found that SGalCer was the best ligand for each rgp120 and HIV-1 isolate tested . Therefore, novel multivalent glycodendrimers, which mimic the carbohydrate clustering reportedly found in lipid rafts, were synthesized based on the carbohydrate moiety of SGalCer . Here we describe the synthesis of a polysulfated galactose functionalized, fifth generation DAB dendrimer (PS Gal 64mer), containing on average two sulfate groups per galactose residue . Its ability to inhibit HIV-1 infection of cultured indicator cells was compared to that of dextran sulfate (DxS), a known, potent, binding inhibitor of HIV-1 . The results indicate that the PS Gal 64mer inhibited infection by the HIV-1 isolates tested as well as DxS . Biofilm Growth and Detachment of Actinobacillus actinomycetemcomitans. Jeffrey B. Kaplan, 2003.The gram-negative, oral bacterium Actinobacillus actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans . When cultured in broth, fresh clinical isolates of A . actinomycetemcomitans form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize the oral cavity and cause disease . We examined the morphology of A . actinomycetemcomitans biofilm colonies grown on glass slides and in polystyrene petri dishes by using light microscopy and scanning and transmission electron microscopy . We found that A . actinomycetemcomitans developed asymmetric, lobed biofilm colonies that displayed complex architectural features, including a layer of densely packed cells on the outside of the colony and nonaggregated cells and large, transparent cavities on the inside of the colony . Mature biofilm colonies released single cells or small clusters of cells into the medium . These released cells adhered to the surface of the culture vessel and formed new colonies, enabling the biofilm to spread . We isolated three transposon insertion mutants which produced biofilm colonies that lacked internal, nonaggregated cells and were unable to release cells into the medium . All three transposon insertions mapped to genes required for the synthesis of the O polysaccharide (O-PS) component of lipopolysaccharide . Plasmids carrying the complementary wild-type genes restored the ability of mutant strains to synthesize O-PS and release cells into the medium . Our findings suggest that A . actinomycetemcomitans biofilm growth and detachment are discrete processes and that biofilm cell detachment evidently involves the formation of nonaggregated cells inside the biofilm colony that are destined for release from the colony . Enhanced Production of Recombinant Proteins in Escherichia coli by Filamentation Suppression. Ki Jun Jeong, 2003.During growth of high-cell-density cultures of Escherichia coli, overproduction of recombinant proteins often results in increased stress response, cell filamentation, and growth cessation . Filamentation of cells consequently lowers final achievable cell concentration and productivity of the target protein . Reported here is a methodology that should prove useful for the enhancement of cell growth and protein productivity by the suppression of cell filamentation . By the coexpression of the E . coli ftsA and ftsZ genes, which encode key proteins in cell division, growth of recombinant strains as well as production of human leptin and human insulin-like growth factor I was improved . Observation of cell morphology revealed that the coexpression of the ftsA and ftsZ genes successfully suppressed filamentation caused by the accumulation of recombinant proteins .
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