Replisome pausing in mutagenesis

E. coli cells containing a temperature-sensitivednaE mutation, in the α-subunit of holoenzyme DNA polymerase III, do not survive at the restrictive temperature. Such cells may survive in the presence of thepcbA1 mutation, an allele of thegyrB gene. Such survival is dependent on an active DNA polymerase I. Evidence indicates that DNA polymerase I interacts directly in the replisome (REP·A). Despite normal survival for cells using thepcbA replication pathway after some type of DNA damage, we have noted a failure of damage-induced mutagenesis. Here we present evidence supporting a model of replisome pausing in cells dependent upon thepcbA replication pathway. The model argues that the (REP·A) complex pauses longer at the site of the lesion, allowing excision repair to occur completely. In the normal replication pathway (REP·E) bypass of the lesion occurs, fixing the mutation.     

Foraging interactions between Wandering Albatrosses Diomedea exulans breeding on Marion Island and long-line fisheries in the southern Indian Ocean

Wandering Albatrosses Diomedea exulans are frequently killed when they attempt to scavenge baited hooks deployed by long-line fishing vessels. We studied the foraging ecology of Wandering Albatrosses breeding on Marion Island in order to assess the scale of interactions with known long-line fishing fleets. During incubation and late chick-rearing, birds foraged further away from the island, in warmer waters, and showed high spatial overlap with areas of intense tuna Thunnus spp. long-line fishing. During early chick-rearing, birds made shorter foraging trips and showed higher spatial overlap with the local Patagonian Toothfish Dissostichus eleginoides long-line fishery. Tracks of birds returning with offal from the Toothfish fishery showed a strong association with positions at which Toothfish long-lines were set and most diet samples taken during this stage contained fishery-related items. Independent of these seasonal differences, females foraged further from the islands and in warmer waters than males. Consequently, female distribution overlapped more with tuna long-line fisheries, whereas males interacted more with the Toothfish long-line fishery. These factors could lead to differences in the survival probabilities of males and females. Non-breeding birds foraged in warmer waters and showed the highest spatial overlap with tuna long-line fishing areas. The foraging distribution of Marion Island birds showed most spatial overlap with birds from the neighbouring Crozet Islands during the late chick-rearing and non-breeding periods. These areas of foraging overlap also coincided with areas of intense tuna long-line fishing south of Africa. As the population trends of Wandering Albatrosses at these two localities are very similar, it is possible that incidental mortality during the periods when these two populations show the highest spatial overlap could be driving these trends.     

Structure function relationships for the IL 2 receptor system. III. Tac protein missing amino acids 102 to 173 (exon 4) is unable to bind IL 2: detection of spliced protein after L cell transfection

High affinity receptors for interleukin 2 (IL 2) contain the Tac protein as one ligand-binding subunit. Localization of the IL 2-binding site on this molecule, as well as localization of the complementary site on IL 2, should provide insight into the design of IL 2 analogs. In this report, we examine the ability of normal and modified Tac protein to bind IL 2 and several antibodies that recognize the native Tac molecule. Using a transient L cell expression system, we have determined that transfection with cDNA-missing Tac exon 4 resulted in expression of spliced protein that had no measurable binding to IL 2 or the monoclonal anti-receptor antibodies, anti-Tac, and 7G7/B6. This protein was detected, however, by rabbit polyclonal antibodies prepared against synthetic Tac peptides. Thus, one or more amino acids encoded by exon 4 is important, either for direct ligand contact or for the proper folding of critical segments of the Tac molecule. In addition, insertion of stop codons at a unique restriction enzyme site near the beginning of exon 5 resulted in cellular secretion of truncated Tac molecules that were capable of binding IL 2, anti-Tac, and 7G7/B6. Amino acids encoded by exons 5 to 8 thus play no critical role in IL 2 binding. The ligand association demonstrated for truncated Tac protein produced by exons 2 to 4 should guide attempts to define the IL 2-binding segment of the Tac molecule.     

Age at maturity in landlocked and anadromous Atlantic salmon parr from two Québec rivers

Synopsis Age at maturity in male Atlantic salmon parr from landlocked populations in the Watshishou and Musquaro Rivers is significantly greater than in anadromous populations from the same rivers. We conclude that high post-smolt mortality in anadromous stocks is conducive to male parr maturity at an early age. We also suggest that the lower proportion of maturing male parr in landlocked stocks may be related to competition among males for mates and the smaller ultimate size of spawning adult landlocked salmon.     

The suppressive effect of gangliosides upon IL 2-dependent proliferation as a function of inhibition of IL 2-receptor association

Gangliosides inhibited the proliferation of mitogen-activated human peripheral blood lymphocytes and the IL 2-dependent growth of murine T cell lines and 5-day-old human PHA lymphoblasts. In the case of the murine cell lines and PHA lymphoblasts, most of the effect of gangliosides could be reversed by the addition of high levels of IL 2. In the case of freshly-stimulated mitogen blasts, however, the ganglioside-induced inhibition could not be reversed by increasing exogenous IL 2 levels. These results indicate that inhibition of proliferation by gangliosides can be divided into IL 2-reversible and IL 2-irreversible mechanisms, the latter of which were predominant during the initial stage of cellular activation. Inclusion of gangliosides in receptor binding assays for radiolabeled IL 2 indicated that the IL 2-reversible mechanism likely involved competition between gangliosides and the cellular receptor for the binding of IL 2. Gangliosides blocked binding of radiolabeled IL 2 to both the high and low affinity forms of the IL 2 receptor, and this effect was most noticeable when the gangliosides and IL 2 were preincubated before addition of the target cells. In contrast, treatment of cells with gangliosides had no effect on the affinity of the cellular IL 2 receptor if the free gangliosides were removed immediately before the binding assay. Gangliosides also blocked the binding of radiolabeled IL 2 to anti-IL 2 antibodies, supporting the notion that their inhibitory effect is mediated via a direct interaction with IL 2. Thus, one major mechanism by which gangliosides block the IL 2-dependent proliferation of activated cells is by the sequestering or inactivation of the IL 2 molecule. This effect is reversible with the addition of excess IL 2, which distinguishes it from other mechanisms of ganglioside-dependent inhibition operating during the cellular activation process.     

In vivo administration of purified human interleukin 2. I. Half-life and immunologic effects of the Jurkat cell line-derived interleukin 2

A total of 12 patients with cancer or the acquired immunodeficiency syndrome have been treated with Jurkat-derived purified human interleukin 2 (IL 2). The toxicity was dose-related and consisted primarily of fever, chills, malaise, and mild reversible hepatic dysfunction. No evidence of clinical efficacy was seen when IL 2 was administered at doses of up to 2000 micrograms by bolus or continuous infusion once a week for 4 wk. No significant chronic immunologic effects (changes in mitogen responsiveness of induction of cytotoxic cells) were demonstrated. IL 2 was measured in the serum of patients, and a half-life of approximately 5 to 7 min was demonstrated with a second component of clearance of 30 to 120 min. Heating the serum at 56 degrees C for 30 min allowed for detection of smaller quantities of IL 2 by removing a serum inhibitor whose effect was seen at dilutions of up to 1/80 in our biologic assay. Sustained levels of IL 2 could be maintained by continuous infusion. Acute effects of IL 2 administration included a rapid decrease in peripheral mononuclear cells with a shift to cells of macrophage lineage and a rapid decrease in total T lymphocytes and T lymphocyte subsets. IL 2 responsiveness of peripheral mononuclear cells decreased within 15 min of IL 2 administration, with a concurrent decrease in the ability to generate lymphokine-activated killer cells. These changes did not recover until 48 hr after IL 2 administration. A rise in serum ACTH and cortisol levels was seen after the administration of 1 to 2 mg of IL 2. Future studies will evaluate the role of larger quantities of recombinant IL 2 given alone or in conjunction with in vitro-generated lymphokine-activated killer cells.