Intermediate mutation frequencies favor evolution of multidrug resistance in Escherichia coli

In studying the interplay between mutation frequencies and antibiotic resistance among Escherichia coli natural isolates, we observed that modest modifications of mutation frequency may significantly influence the evolution of antibiotic resistance. The strains having intermediate mutation frequencies have significantly more antibiotic resistances than strains having low and high mutation frequencies.     

Transport of groundwater-borne nutrients from watersheds and their effects on coastal waters

Anthropogenic activities on coastal watersheds increase nutrient concentrations of groundwater. As groundwater travels downslope it transports these nutrients toward the adjoining coastal water. The resulting nutrient loading rates can be significant because nutrient concentrations in coastal groundwaters may be several orders of magnitude greater than those of receiving coastal waters. Groundwater-borne nutrients are most subject to active biogeochemical transformations as they course through the upper 1 m or so of bottom sediments. There conditions favor anaerobic processes such as denitrification, as well as other mechanisms that either sequester or release nutrients. The relative importance of advective vs. regenerative pathways of nutrient supply may result in widely different rates of release of nutrients from sediments. The relative activity of denitrifiers also may alter the ratio of N to P released to overlying waters, and hence affect which nutrient limits growth of producers. The consequences of nutrient (particularly nitrate) loading include somewhat elevated nutrient concentrations in the watercolumn, increased growth of macroalgae and phytoplankton, reduction of seagrass beds, and reductions of the associated fauna. The decline in animals occurs because of habitat changes and because of the increased frequency of anoxic events prompted by the characteristically high respiration rates found in enriched waters.     

The effect of feeding by mud snails, Ilyanassa obsoleta (Say), on the structure and metabolism of a laboratory benthic algal community

Eastern mud snails, Ilyanassa obsoleta (Say), in densities of 0, 80, and 160 snails · m^?2 were placed in flow-through laboratory microcosms containing 5 cm of frozen and sieved sediments. Other microcosms were raked once daily to a depth of 10 mm. All these containers were incubated for 5 wk and regularly sampled for plant pigments and light and dark transfer of oxygen and carbon dioxide. Feeding at the low density significantly increased chlorophyll standing stock. Respiration and gross photosynthesis increased by an even greater percentage compared to ungrazed controls. Standing stocks of algal pigments, respiration, and photosynthesis were depressed in microcosms which received the 160-snail or raking treatments.The dominant benthic algae in the containers were pennate diatoms. Grazed containers contained a larger percentage of non-motile as compared to motile forms.Sediments fertilized with ammonium at a rate equivalent to excretion of six snails, showed increased chlorophyll content equal to the 80-snail treatment. Daily raking inhibited this effect.We conclude that low densities of Ilyanassa obsoleta stimulate algal growth by accelerating nitrogen cycling and selectively removing specific components of the diatom community. At high snail densities these effects are overwhelmed by overgrazing and sediment stirring.     

PTIP/Swift is required for efficient PCNA ubiquitination in response to DNA damage

Monoubiquitination of proliferating cell nuclear antigen (PCNA) enables translesion synthesis (TLS) by specialized DNA polymerases to replicate past damaged DNA. We have studied PCNA modification and chromatin recruitment of TLS polymerases in Xenopus egg extracts and mammalian cells. We show that Xenopus PCNA becomes ubiquitinated and sumoylated after replication stress induced by UV or aphidicolin. Under these conditions the TLS polymerase eta was recruited to chromatin and also became monoubiquitinated. PTIP/Swift is an adaptor protein for the ATM/ATR kinases. Immunodepletion of PTIP/Swift from Xenopus extracts prevented efficient PCNA ubiquitination and polymerase eta recruitment to chromatin during replicative stress. In addition to PCNA ubiquitination, efficient polymerase eta recruitment to chromatin also required ATR kinase activity. We also show that PTIP depletion from mammalian cells by RNAi reduced PCNA ubiquitination in response to DNA damage, and also decreased the recruitment to chromatin of polymerase eta and the recombination protein Rad51. Our results suggest that PTIP/Swift is an important new regulator of DNA damage avoidance in metazoans.     

Preferential Localization of Tyrosine-Phosphorylated Paxillin in Focal Adhesions

Focal adhesions are sites for integrin-mediated attachment of cultured cells to the extracellular matrix. Localization studies have shown that focal adhesions can be stained by antiphosphotyrosine antibodies, but the role of tyrosine-phosphorylated proteins in focal adhesions is not known. By using ventral plasma membranes prepared from chicken embryo fibroblasts spread on the substrate, we present evidence for the preferential localization of a minor pool of tyrosine-phosphorylated paxillin in focal adhesions. Ventral plasma membranes showed an enrichment in β1-integrins, and in several tyrosine-phosphorylated polypeptides, while focal adhesion proteins like vinculin and paxillin, although localized to focal adhesions in ventral plasma membranes, were not particularly enriched in these preparations compared to whole cell lysates. Biochemical and morphological analysis of ventral plasma membranes showed a dramatic increase in the level of tyrosine-phosphorylation of the pool of paxillin localized to the adhesive sites, when compared to the paxillin present in whole cell lysates. The observed preferential localization of tyrosine-phosphorylated paxillin to focal adhesions may represent a general mechanism to compartmentalize focal adhesion components from large non-phosphorylated, cytosolic pools.     

Evaluation of phage display system and leech-derived tryptase inhibitor as a tool for understanding the serine proteinase specificities

A small combinatorial library of LDTI mutants (5.2 x 10(4)) restricted to the P1-P4' positions of the reactive site was displayed on the pCANTAB 5E phagemid, and LDTI fusion phages were produced and selected for potent neutrophil elastase and plasmin inhibitors. Strong fusion phage binders were analyzed by ELISA on enzyme-coated microtiter plates and the positive phages had their DNA sequenced. The LDTI variants: 29E (K8A, I9A, L10F, and K11F) and 19E (K8A, K11Q, and P12Y) for elastase and 2Pl (K11W and P12N), 8Pl (I9V, K11W, and P12E), and 10Pl (I9T, K11L, and P12L) for plasmin were produced with a Saccharomyces cerevisiae expression system. New strong elastase and plasmin inhibitors were 29E and 2Pl, respectively. LDTI-29E was a potent and specific neutrophil elastase inhibitor K(i) =0.5 nM), affecting no other tested enzymes. LDTI-2Pl was the strongest plasmin inhibitor ( K(i) =1.7nM) in the LDTI mutant library. This approach allowed selection of new specific serine proteinase inhibitors for neutrophil elastase and plasmin (a thrombin inhibitor variant was previously described), from a unique template molecule, LDTI, a Kazal type one domain inhibitor, by only 2-4 amino acid replacements. Our data validate this small LDTI combinatorial library as a tool to generate specific serine proteinase inhibitors suitable for drug design and enzyme-inhibitor interaction studies.     

Modulation of cell cycle progression and of antibody production in mouse hybridomas by a nucleotide analogue

The nucleotide analogue 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) has been identified as a powerful antiproliferative substance when acting on hybridoma cells. In the range of 10 nM to 100 nM concentrations this agent reduces cell growth rate, while its apoptosis-inducing activity is marginal. Marked induction of apoptosis can be observed at micromolar and higher order concentrations. In PMEG-supplemented media the cell cycle progression is perturbed, the flow-cytometric DNA profile shows a higher proportion of cells in the S and G2/M phases of the cell cycle. Concomitantly with the reduction of the growth rate, the specific monoclonal antibody production rate may rise by 20–27%. Addition of PMEG at the end of the exponential phase of a batch culture results in an enhancement of the final monoclonal antibody concentration. This revised version was published online in August 2006 with corrections to the Cover Date.