Advances in cytochemical methods for detection of apoptosis.

In 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. (J Histochem Cytochem 49:821-832, 2001)

Do non-insulin-dependent diabetes mellitus (NIDDM) and insulin-dependent diabetes mellitus (IDDM) share genetic susceptibility loci? An analysis of putative IDDM susceptibility regions in familial NIDDM.

Non-insulin-dependent diabetes mellitus (NIDDM) has been viewed as genetically and physiologically distinct from insulin-dependent diabetes mellitus (IDDM), yet many of the recently suggested IDDM susceptibility loci are likely to increase the risk of diabetes through nonautoimmune mechanisms. To test the hypothesis that the IDDM susceptibility loci include important NIDDM susceptibility loci, we tested the linkage of 14 putative susceptibility regions with NIDDM among families and sibling pairs of Northern European descent. All regions were tested with highly informative microsatellite (simple tandem repeat) polymorphisms in up to 166 affected individuals from 42 families using both parametric and nonparametric methods (149 pairs for sibling pair analyses). We found no evidence for linkage to the majority of loci, including loci that appeared to be linked to IDDM in more than one study. We report some evidence for shared susceptibility for regions on chromosomes 1, 2, and 6. The best evidence based on multilocus affected pedigree member (APM) analysis of markers near D1S191 suggested linkage at P value .0001. This region has not yet been confirmed as an IDDM locus, and our analyses could represent a false-positive result. The role of these three regions will only be clarified by testing in additional families. In combination with other investigations in our laboratory for chromosome 11 susceptibility regions, our data generally do not provide convincing evidence that IDDM and NIDDM share common genetic factors among families of Northern European descent with ascertainment of two or more NIDDM siblings.