Evidence for multiple lytic pathways used by cytotoxic T lymphocytes

Previous data generated by ourselves and others questioned the role of degranulation as a mechanism to explain CTL-mediated cytotoxicity. In this report we examine this tissue in greater depth. CTL-mediated lysis was probed with three different inhibitors. 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene inhibits degranulation in a wide range of cell types, including CTL. EGTA, through chelation of Ca2+, also inhibits degranulation processes in CTL, and would inhibit other events or processes dependent on extracellular Ca2+. We also used prolonged exposure to PMA to exhaust PKC activity in CTL. Using these inhibitors, we have defined three pathways of lysis used by CTL. One pathway requires Ca2+, is PMA sensitive, but does not depend on degranulation. The second pathway is independent of Ca2+, is not PMA sensitive, and also does not depend on degranulation. All primary CTL and cloned CTL lyse most target cells via pathway I. However, when confronted with certain target cells (which we have referred to previously as Ca2+-independent target cells), pathway II is induced. When pathway II is induced, pathway I apparently shuts down. We show here that pathway II does not depend on protein synthesis, and that it also leads to DNA solubilization in target cells. A limited number of cloned CTL use pathways I and II as just described, but use in addition, and simultaneously, a third pathway that appears to involve degranulation. This pathway is seen irregularly in most CTL clones, and may be influenced by levels of IL-2 in the culture medium.     

Covalent interaction of L-2-amino-4-oxo-5-chloropentanoic acid with rat renal phosphate-dependent glutaminase. Evidence for a specific glutamate binding site and of subunit heterogeneity

Rat renal phosphate-dependent glutaminase is rapidly inactivated by incubating with L-2-amino-4-oxo-5-chloropentanoic scid. Concentrations of phosphate, which increase the glutaminase activity, decrease the rate of inactivation by chloroketone. In addition, inactivation is not blocked by glutamine. Instead, glutamate was shown to specifically reduce the rate of chloroketone inactivation. Upon sodium lauryl sulfate-polyacrylamide gel electrophoresis, the purified glutaminase preparation exhibits at least five protein staining bands which range in molecular weight from 57,000 to 75,000. Studies with 14C-labeled chloroketone indicate that this reagent reacts with each of these peptides. The mean stoichiometry of binding was calculated to be 1.3 mol/mol of enzyme. Therefore, these results indicate that the glutaminase may contain a specific site for binding glutamate and that the purified enzyme consists of a series of related peptides which may have resulted from partial proteolysis.     

Observations on lesser-known flatworms: temnocephala

Temnocephala novae-zealandie, a flatworm epizoic on crayfish, was examined by light and electron microscopy to investigate the variation within rhabdocoel turbellarians and to provide information on the possible structural modifications in the evolution of parasitism. The sucker is clearly glandular; the tentacular glands are eosinophilic and at the surface store and often release mucus as a coiled thread; the epidermis is clearly not reduced in structure and contains septate junctions in the anterior portion. Light microscope studies documented the presence of ten pair of paranephrocytes (athrocytes). In the laboratory, egg deposition, survival on and apart from the host and another crayfish, and behavior during the host molt were observed.     

Parameters of brown-headed cowbird Molothrus ater egg discrimination in warbling vireos Vireo gilvus

We examined which egg parameters warbling vireos Vireo gilvus use to discriminate brown-headed cowbird Molothrus ater eggs and, by comparing our results to other studies, tested the prediction that ejecter species with eggs more similar in appearance to cowbird eggs will be less tolerant of foreign eggs. In addition, we tested the hypothesis that egg characteristics influence the cost of ejection and probability of committing ejection errors. Warbling vireos ejected 100% of eggs with a cowbird spot pattern and only spot pattern significantly influenced the probability of ejecting a foreign egg, whereas size and nest stage did not. Foreign eggs that differed in two parameters were not ejected significantly more than those that differed in one parameter. Thus, warbling vireos appear to be less tolerant of foreign eggs than species with eggs more divergent from cowbird eggs. There was no significant difference in the number of vireo eggs that were damaged when foreign eggs of different sizes and spot patterns were ejected, which is counter to the assumptions of the evolutionary equilibrium hypothesis. Similarly, foreign egg characteristics did not significantly influence the probability of ejection errors. Finally, egg discrimination in warbling vireos appears to have evolved directly to counter cowbird parasitism because all conspecific eggs switched into their nests were accepted.     

Small hosts infrequently disrupt laying by Brown-headed Cowbirds and Bronzed Cowbirds

ABSTRACT Brood parasites often must overcome host defenses that may include behaviors that serve other functions, such as deterrence of predators and nest attendance during laying and incubation. Host use by brood parasites may also be influenced by competitors in areas where more than one parasitic species occurs. We identified the degree to which behavior of potential hosts and potential competitors affected laying by Brown‐headed Cowbirds (Molothrus ater) and Bronzed Cowbirds (M. aeneus) at a site in south Texas where they co‐occur. We watched potential host nests during the presunrise period to record cowbird laying and document nest visitation, laying, cowbird‐host encounters, and nest attentiveness by hosts. Hosts were frequently at their nests when cowbirds laid eggs (83% of 121 watches among nests of five host species) and cowbirds regularly encountered hosts (43–74% and 40–77% of watches per species of host for Brown‐headed and Bronzed cowbirds, respectively). Host nest defense infrequently interfered with cowbird laying and cowbirds rarely interacted with one another during laying. Overall, 12% of the 42 cowbird laying attempts that elicited host nest defense failed, resulting in cowbird eggs either laid atop hosts as they sat in nests or laid outside the nest cup. We clearly documented that relatively small hosts can thwart parasitism by cowbirds. Thus, the potential for successful defense of nests should be considered when assessing the evolution of behaviors to deter the removal of host eggs by cowbirds and mechanisms leading to nest abandonment. Regarding the latter, the presence of a cowbird at a nest would be a poor indicator for parasitism as some laying attempts were thwarted and unparasitized broods were reared at those nests. Despite the potential for nest defense to affect host use by cowbirds, we did not detect an effect of nest defense. Because most host defense was ineffective, we examined hypotheses for the timing of cowbird laying and host nest attendance. Our analysis of time of day of laying by Brown‐headed Cowbirds at our site and data compiled from the literature suggests that laying time is best predicted by the time of civil twilight (first light) rather than sunrise.