Bacteriophage production by doubly lysogenic Corynebacterium diphtheriae.

Parental and recombinant phage production by tandem, double lysogens of Corynebacterium diphtheriae was studied in strains in which the coupling of prophage markers and the order of prophage was established. The results from studies of mass lysates and single bursts showed that the recombinant class of phage, designated R1, was predominant in UV-induced lysates followed by the parental, P1 class and to a lesser extent the P2 and R2 classes. Single bursts of UV-treated cells contained phage from one to all four of the phage classes, and this appeared to reflect the action of two excision processes. The data indicate that recombinant phages R1 and R2 are formed by a process of general recombinational excision and that this is the primary event leading to phage production in both UV-irradiated and spontaneously induced double lysogens. This process, which depends on exchange between homologous genes and is reciprocal, accounts for the excision of R1 phage from the host chromosome. A second excision process, probably site-specific excision, also occurs in many of the same cells and accounts for the excision of P1, P2, and R2 phages. The significance of these results for the spread of toxinogenicity in strains of C. diphtheriae is discussed.     

Isolation and characterization of tox mutants of corynebacteriophage beta.

Seventeen nontoxinogenic (tox) mutants of corynebacteriophage beta have been isolated by using a tissue culture screening technique. The mutants fall into four major classes. Two of the classes, I and II, appear to contain missense and nonsense mutants, respectively. However, classes III and IV have not been previously described. Class III mutants produce two proteins (CRMs) seriologically related to diphtheria toxin, but efforts to demonstrate the presence of more than one tox gene have been successful. Class IV mutants are phenotypically CRM-, failing to produce any detectable protein serologically related to diphtheria toxin. Genetic studies indicate that the mutations in class IV strains are not in a gene distinct form the structural gene for toxin, and that the CRM- strains retain at least a portion of that gene. A natural phage isolate, gamma, behaves in a completely parallel fashion to the class IV mutants. The production of tox+ recombinants through recombination of various pairs of tox phage mutants has been demonstrated. The implications of these findings for the natural history of diphtheria are discussed.     

Bottom-Up Forces Drive Increases in the Abundance of Large Daphnids in Four Small Lakes Stocked with Rainbow Trout (Oncorhynchus mykiss), Interior British Columbia,Canada

Ecosystems - The introduction of salmonids into lakes of western North America for sport fishing is a widespread phenomenon. While numerous investigations have documented cascading trophic...     

Maximizing antibody production in a targeted integration host by optimization of subunit gene dosage and position

Historically, therapeutic protein production in Chinese hamster ovary (CHO) cells has been accomplished by random integration (RI) of expression plasmids into the host cell genome. More recently, the development of targeted integration (TI) host cells has allowed for recombination of plasmid DNA into a predetermined genomic locus, eliminating one contributor to clone-to-clone variability. In this study, a TI host capable of simultaneously integrating two plasmids at the same genomic site was used to assess the effect of antibody heavy chain and light chain gene dosage on antibody productivity. Our results showed that increasing antibody gene copy number can increase specific productivity, but with diminishing returns as more antibody genes are added to the same TI locus. Random integration of additional antibody DNA copies in to a targeted integration cell line showed a further increase in specific productivity, suggesting that targeting additional genomic sites for gene integration may be beneficial. Additionally, the position of antibody genes in the two plasmids was observed to have a strong effect on antibody expression level. These findings shed light on vector design to maximize production of conventional antibodies or tune expression for proper assembly of complex or bispecific antibodies in a TI system.     

The K+/H+ antiporter LeNHX2 increases salt tolerance by improving K+ homeostasis in transgenic tomato

The endosomal LeNHX2 ion transporter exchanges H⁺ with K⁺ and, to lesser extent, Na⁺. Here, we investigated the response to NaCl supply and K⁺ deprivation in transgenic tomato (Solanum lycopersicum L.) overexpressing LeNHX2 and show that transformed tomato plants grew better in saline conditions than untransformed controls, whereas in the absence of K⁺ the opposite was found. Analysis of mineral composition showed a higher K⁺ content in roots, shoots and xylem sap of transgenic plants and no differences in Na⁺ content between transgenic and untransformed plants grown either in the presence or the absence of 120 mm NaCl. Transgenic plants showed higher Na⁺/H⁺ and, above all, K⁺/H⁺ transport activity in root intracellular membrane vesicles. Under K⁺ limiting conditions, transgenic plants enhanced root expression of the high‐affinity K⁺ uptake system HAK5 compared to untransformed controls. Furthermore, tomato overexpressing LeNHX2 showed twofold higher K⁺ depletion rates and half cytosolic K⁺ activity than untransformed controls. Under NaCl stress, transgenic plants showed higher uptake velocity for K⁺ and lower cytosolic K⁺ activity than untransformed plants. These results indicate the fundamental role of K⁺ homeostasis in the better performance of LeNHX2 overexpressing tomato under NaCl stress.     

Keratinocyte galvanotaxis in combined DC and AC electric fields supports an electromechanical transduction sensing mechanism

Sedentary keratinocytes at the edge of a skin wound migrate into the wound, guided by the generation of an endogenous electric field (EF) generated by the collapse of the transepithelial potential. The center of the wound quickly becomes more negative than the surrounding tissue and remains the cathode of the endogenous EF until the wound is completely re‐epithelialized. This endogenous guidance cue can be studied in vitro. When placed in a direct current (DC) EF of physiological strength, 100 V/m, keratinocytes migrate directionally toward the cathode in a process known as galvanotaxis. Although a number of membrane‐bound (e.g., epidermal growth factor receptor (EGFR), integrins) and cytosolic proteins (cAMP, ERK, PI3K) are known to play a role in the downstream signaling mechanisms underpinning galvanotaxis, the initial sensing mechanism for this response is not understood. To investigate the EF sensor, we studied the migration of keratinocytes in a DC EF of 100 V/m, alternating current (AC) EFs of 40 V/m at either 1.6 or 160 Hz, and combinations of DC and AC EFs. In the AC EFs alone, keratinocytes migrated randomly. The 1.6 Hz AC EF combined with the DC EF suppressed the direction of migration but had no effect on speed. In contrast, the 160 Hz AC EF combined with the DC EF did not affect the direction of migration but increased the migration speed compared to the DC EF alone. These results can be understood in terms of an electromechanical transduction model, but not an electrodiffusion/osmosis or a voltage‐gated channel model. Bioelectromagnetics 34:85–94, 2013. © 2012 Wiley Periodicals, Inc.     

Imidazo[4,5-b]pyridine inhibitors of B-Raf kinase

This Letter details the synthesis and evaluation of imidazo[4,5-b]pyridines as inhibitors of B-Raf kinase. These compounds bind in a DFG-in, αC-helix out conformation of B-Raf, which is a binding mode associated with significant kinase selectivity. Structure–activity relationship studies involved optimization of the ATP-cleft binding region of these molecules, and led to compound 23, an inhibitor with excellent enzyme/cell potency, and kinase selectivity.