S U M M A R YIn an earlier article from this laboratory, the current methods developed to detect apoptosis in cells and tissues were highlighted, along with the challenges in their interpretation. Recent discoveries concerning the underlying biochemical mechanisms of apoptotic effector pathways have made possible further assays that allow a more direct measure of the activation of the apoptotic machinery in cells. This article summarizes some of these newer methods and extends the interpretation of the more classical assays of apoptosis in a defined cell system. We present data in KB and PC3 cell model culture systems induced to undergo apoptosis by the plant toxin ricin. Using a modified in situ nick translation assay (ISNT) with either Bodipy or BUdR labeling, we confirm that most cells showing altered nuclear morphology do not show reactivity with this assay until very late in the apoptotic process. We also show that only a minority of cells label with fluorescent annexin V during apoptosis but that apoptotic cells continue to internalize material from the cell surface through endocytosis after becoming reactive with annexin V. In addition, we describe the utility of a prototype of new assays for caspase substrate cleavage products, the detection of cleaved cytokeratin 18. It is these newer cleavage product assays that perhaps hold the greatest promise for specific detection of apoptosis in cells either in cell culture or in intact tissues. T he regulation of apoptosis has gained central importance in many aspects of biology, including studies of embryonic development, the pathogenesis of disease, and the response of cells to therapy (Wyllie 1992). The accurate detection of apoptosis has therefore become crucial to these studies. Beginning with the initial observations of changes in nuclear morphology that distinguished apoptosis from necrotic cell death, the field of cell death research has rapidly expanded, generating a wealth of knowledge about the genetic and biochemical pathways that control this process (reviewed in Green 2000). Several assays for detection of apoptosis were also developed during this past decade, some of which were applicable to cytochemical analysis. However, the interpretation and specificity of these assays, especially the TUNEL assay for DNA strand breaks, have been points of controversy (Allen et al. 1997). These issues have been presented in a previous article from this laboratory, in which we reviewed the available methods for apoptosis detection (Willingham 1999). The present article builds on this prior review, presents some limited new data on modifications of older methods, and reviews some newer methods for apoptosis detection. Figure 1 summarizes a small portion of the apoptosis regulation pathways that have been recently delineated, and it is clear that this new knowledge can be the starting point for the design of more specific assays of apoptosis. Several new assays have recently appeared that depend on newly discovered changes at the molecular level. These changes are ...

Beginning in the mid-1980s, the Laurentian Great Lakes underwent successive invasions by Ponto-Caspian species. We quantified major changes in the diversity and relative abundance of pre-invasion benthic macroinvertebrates at the same study site in southwestern Lake Ontario from 1983-2014. The zebra mussel Dreissena polymorpha Pallas arrived at the study site before 1991, the quagga mussel Dreissena rostriformis bugensis Andrusov and the amphipod Echinogammarus ischnus Stebbing arrived before 1999, and the Round Goby Neogobius melanostomus Pallas arrived about 2004. The macroinvertebrate community in 2014 was very different from 3 earlier communities in 1983, 1991, and 1999. In 2014, pulmonate and prosobranch snails and sphaeriid bivalves were absent, D. r. bugensis replaced D. polymorpha, E. ischnus replaced Gammarus fasciatus Say as the dominant amphipod, and a previously diverse community of benthic fish was replaced by abundant N. melanostomus. From 1983 to 1999, the relative abundance of prosobranchs and pulmonates declined 10-fold and rose 2-fold, respectively. From 1991 to 2014, the relative abundance of oligochaetes and chironomids increased 32-and 78-fold, respectively. The shifts we report probably are attributable to nutrient enrichment of the nearshore of Lake Ontario during the 1990s leading to a thick carpet of macroalgae, a change in the base of the benthic food web from dressenid feces and pseudofeces to macroalgal detritus, and predation by N. melanostomus on snails.

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