A B S T R A C T The contractility of platelets has been attributed to an actomyosin-like protein which has been well defined on a physicochemical basis. Moreover, platelets contain ±80 A filaments which resemble actin filaments in smooth muscle. Studies were undertaken on human and bovine platelets to better define the morphologic structures which may subserve this contractile function. In order to identify actin, the ability of the filaments to react with heavy meromyosin (HMM) was tested. Accordingly, platelets were glycerinated and treated with HMM. In addition, platelet actin was extracted, reacted with HMM, and examined by negative staining. In both instances typical arrowhead structures with clearly defined polarity and a periodicity of ±360 A formed. As is the case with purified muscle actin, the complexes were dissociable with Mg-ATP. The formation of myosin-like filaments was observed when osmotically shocked platelets were incubated with MgCla and excess ATP. These "thick" filaments measured 250-300 A in width, tapered at both ends and often occurred in clumps. They resembled aggregates of thick filaments described in contracted smooth muscle. Extraction of platelets by methods suitable for the demonstration of myosin showed filaments with an average length of 0.3 /, a smooth shaft, and frayed or bulbous ends. These appeared identical to those seen in synthetically prepared myosin of striated muscle. It is suggested that the filaments described here represent the actin and myosin of platelets.
Ultrastructural cytochemistry of natural killer cells enriched by Percoll gradient centrifugation showed them to possess arylsulfatase (aryl-sulfate sulfohydrolase, EC 3.1.6.1). The enzyme was located in vesicles, granules, and the parallel tubular arrays, organelles characteristic for cytotoxic lymphocytes. Biochemically, peak enzyme activity correlated with the Percoll fractions containing cells with cytotoxicity for melanoma target cells. Treatment of natural killer cells with Na2SO4, a competitive inhibitor of arylsulfatase, suppressed cytotoxicity by almost 50%. Electron microscopy of effector-target cell conjugates, which had been permitted to incubate for only 30 min, disclosed numerous arylsulfatase-positive sites at the points of contact between the effector/target cell membranes. Thus, the enzyme was translocated to the surface before lysis of the target cell was morphologically evident. It is postulated that the parallel tubular arrays play a role in this translocation and that arylsulfatase may function in the degradation of cerebroside sulfate ester components of the target cell membrane to initiate the lytic event.A small subpopulation of peripheral blood lymphocytes is able to kill tumor cells in vivo and in vitro without any prior exposure to the neoplasm. The cells endowed with such ability have been called natural killer (NK) cells and their properties are fairly well defined (1, 2). They are recognized as nonadherent, nonphagocytic, large granular lymphocytes that have receptors for the Fc portion of IgG, a weak affinity for sheep erythrocytes, and an ultrastructural marker, the parallel tubular array (PTA) (3, 4). Monoclonal antisera specific for some of their surface antigens also have been developed (5). It is not known how NK cells select their targets and by what mechanism they inflict their lethal effect. However, physical contact between the effector and target cell appears to be necessary for tumor cell lysis to occur. The remarkable interdigitation between the surface membranes of the conjugated cells also has been illustrated repeatedly (4,6,7). However, because it has not been possible to resolve any membrane damage, even on electron microscopy, it is generally assumed that the biochemical alterations that lead to swelling and subsequent lysis of the target cells are beyond morphologic resolution. This has led to biochemical studies intended to show that proteinases associated with NK cells or secreted by them may be responsible for target cell lysis. Indeed, inhibitors of serine proteinases partially inhibit the NK cell effect (4, 6, 8). In our own hands, inhibitors of trypsin-like enzymes and serine esterases have substantially impaired NK cell function (4). A role for free oxygen radicals generated on cell contact has been suggested by others (9, 10). In addition, the translocation of acid hydrolases has been proposed as a possible mechanism involved in cytotoxicity (6, 7). A group of enzymes not heretofore considered to play a role in target cell lysis are the arylsulfa...
There has been a need to easily quantitate the incidence of eosinophil colonies within soft agar cultures. This has been realized by layering of the agar with benzidine dihydrochloride that permits detection of peroxidase activity in cells. Eosinophil colonies can be specifically identified by the addition to the substrate of potassium cyanide, an inhibitor of enzyme activity in neutrophils and monocytes. The enumeration of eosinophil colonies can be accomplished by scanning fresh or embedded cultures with low power magnification.
SYNOPSIS. Changes accompanying aging in light‐grown stationary cultures of Ochromonas danica were examined cytochemically. Succinate dehydrogenase activity increased during the log phase and decreased steadily during stationary and later phases. Acid phosphatase, alkaline phosphatase and lipase activities increased during the several phases of growth, as did accumulation of lipid. These results imply loss of mitochondrial activity and a gain in lysosomal activity with aging of the cell population. Alkaline phosphatase, widely distributed in animals and believed absent from most photosynthetic organisms and bacteria, is here reported in the photosynthetic genus Ochromonas.
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