Standard quantitative assays for apoptosis

The term apoptosis refers to a peculiar morphology of cell death. It is of special interest because it can be triggered physiologically (and pathologically), and it is regulated by the actions of specific gene products. Therefore, it can in principle be activated and suppressed by medical intervention. It thus is often important to determine whether cells are dying by apoptosis (or its less regulated counterpart, necrosis) and also to quantity the effect in a population of cells. Here the classic methods of apoptosis quantitation are described; they will be of particular use to those whose laboratories are set up for standard microscopical and biochemical techniques, who do apoptosis assays infrequently but wish them to be widely accepted and reproducible. A simple microscopic observation, using blue light illumination and a pair of fluorescent dyes, is recommended for most applications.     

Applications of new saturation transfer electron paramagnetic resonance methodology to the rotational dynamics of the Ca-ATPase in sarcoplasmic reticulum membranes.

The presence of small amounts of weakly immobilized probes can result in large systematic errors in the measurement of correlation times (tau r) from saturation transfer EPR spectra. However, we have recently developed experimental methodology to minimize these errors (Squier and Thomas, Biophys. J., 49:921-935). In the present study we have applied this methodology to the measurement of the rotational motion of the Ca-ATPase in sarcoplasmic reticulum. This analysis involves the estimate of tau r from line-shape parameters (spectral line-height ratios) and intensity parameters (spectral integral), coupled with digital subtractions to remove spectral components corresponding to weakly immobilized probes. We have analyzed the ST-EPR spectra of the Ca-ATPase over a range of temperatures and find that, unlike line-shape parameters, intensity parameters are little affected by the subtraction of the weakly immobilized spectral component (W). Thus, tau r values from intensity parameters are a more reliable measurement of rotational motion. As reported previously, an analysis with line-shape parameters yields a nonlinear Arrhenius plot of protein mobility. However, the plot is linear when intensity parameters or corrected spectra are used, consistent with the theory for the hydrodynamic properties of a membrane protein of unchanging size and shape in a fluid bilayer. An analysis with line-shape parameters yields different effective tau r values in different spectral regions, and these tau r values are temperature-dependent. However, correction of spectra for W yields temperature-independent tau r ratios, indicating that the motional anisotropy is temperature-independent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Commentary: moral growth in medical students

Knight has shown how the moral growth of medical students involves a spiritual journey. He may, however, present too sanguine a portrayal of the extent to which the medical education environment promotes this moral and spiritual growth. Medical school may indeed be more abusive than supportive. Admitting more women to medical school and teaching more humanities courses, while worthwhile, will not necessarily promote the goals that Knight appropriately advocates.     

Human alveolar macrophage fibronectin: synthesis, secretion, and ultrastructural localization during gelatin-coated latex particle binding

Human pulmonary alveolar macrophages synthesized and secreted several characteristic high molecular weight proteins for at least 7 d in vitro. Immunoprecipitates of medium and cell lysates from metabolically labeled cultures with specific anti-human plasma fibronectin IgG contained one major labeled polypeptide of molecular weight 440,000 (unreduced) or 220,000 (reduced). An identical polypeptide in conditioned medium from radiolabeled macrophages bound specifically to gelatin-Sepharose, demonstrating that alveolar macrophages synthesized and secreted a molecule immunologically and functionally similar to fibronectin. Fibronectin was the major newly synthesized and secreted polypeptide of freshly harvested alveolar macrophages. Pulse-chase experiments revealed that newly synthesized fibronectin was rapidly secreted into medium, approximately 50 percent appearing by 1 h and 80 percent by 8 h. Immunoperoxidase staining using antifibronectin F(ab’)(2)-peroxidase conjugates revealed the majority of immunoreactive fibronectin to be intracellular, localized to endoplasmic reticulum and Golgi apparatus. No extracellular matrix fibronectin was visualized, and cell surface staining was rarely seen, usually appearing only at sites where cells were closely apposed and not at sites of macrophage-substrate attachment. Similar immunostaining of fibroblast cultures revealed cell surface-associated fibrillar fibronectin. Ultrastructural localization of fibronectin during binding and phagocytosis of gelatin-coated and plain latex particles revealed fibronectin only on gelatin-latex beads and at their cell binding sites. Neigher plain latex beads nor their cell membrane binding sites stained for fibronectin. These results demonstrate that fibronectin is a major product of human alveolar macrophages, is rapidly secreted, and is localized at cell membrane binding sites for gelatin-coated particles. In view of the known binding properties of fibronectin, it may serve as an endogenous opsonic factor promoting the binding of staphylococcus, denatured collagen, fibrin, or other macromolecules to macrophages in the lower respiratory tract.     

Phylogenetic screening of the human genome: identification of differentially hybridizing repetitive sequence families

The phi-screen, a method of phylogenetic screening, can be employed to detect repetitive sequence families that differentially hybridize between closely related species. Such differences may involve sequence divergence or variations in copy number, including total presence versus absence of a family of repeated DNA. We present the results of a phi-screen comparing the human genome to that of the prosimian, Galago crassicaudatus. Three human repetitive families that are divergent or not present in galago have been detected. One of these families is described in detail; it is similar among the anthropoids but is present in a lower copy number and/or divergent form in prosimians. The family is clearly related to the transposon-like human element (THE) described by Paulson et al. (1985). THEs have long terminal repeats reminiscent of retroviruses but are unique in that they have no sequence similarity to known mammalian retroviruses. The sequence of a solo long terminal repeat, found unassociated with THE internal sequence, is presented. This family member, THE p2, is bordered by a 5-bp target-site repeat and is interrupted by the insertion of an Alu element. A solo THE element sequenced by Wiginton et al. (1986) contains an insertion of Alu at precisely the same position as does THE p2.