John W. Greenawalt

Preparation and characterization of mitochondria and submitochondrial particles of rat liver and liver-derived tissues.

Publisher Summary This chapter discusses techniques for the preparation and characterization of mitochondrial and submitochondrial fractions from liver and liver-derived tissues. The chapter represents the most detailed account of preparative procedures available for mitochondria and submitochondrial particles of rat liver. The chapter initially summarizes several methods for isolating rat liver mitochondria. Some of these methods yield type-1 mitochondria (partially purified but intact), and some yield type-2 mitochondria (highly purified). The two most common tests used to determine the quality of mitochondrial and submitochondrial preparations involve an examination of their respiratory characteristics in a small chamber equipped with an oxygen electrode, and an examination of their structural and morphological features under the electron microscope. The chapter presents flow diagrams that include many of the methods to obtain an overall view of a given procedure. The methodology involved in preparing mitochondria for evaluation in the electron microscope is summarized here, together with a few statements about what each step involves and why it is carried out.

Defective T particles of vesicular stomatitis virus: I. Preparation, morphology, and some biologic properties

Abstract Infection of chick embryo cells with vesicular stomatitis virus under conditions of high multiplicity and serial undiluted passage (VSV up ) results in two kinds of progeny: a low yield of infectious bullet-shaped (B) particles and a high yield of noninfectious (T) particles. The smaller T particles can be partially purified by rate zonal centrifugation in sucrose gradients and readily identified by electron microscopy. T particles appear almost spherical with a diameter of ~65 mμ compared with the typically cylindrical B particles with dimensions of ~65 × 180 mμ. Despite this difference in size, T is strikingly similar to B in its ultrastructure, buoyant density, antigenicity, incorporation of uridine- 3 H during growth, and capacity to inhibit cellular RNA and interferon synthesis. The data strongly suggest that T is a distinct, truncated form of B which contains only a portion of the VSV genome. The accompanying paper provides proof that T is the physical equivalent of the transmissible interfering component of Cooper and Bellett (1959) .

Ultrastructure of striated inclusions in Neurospora

Cytoplasmic and intranuclear bodies appearing as bundles of rodlike structures have been observed in cells of Neurospora crassa. These striated bodies may lie in the cytoplasm, in the nucleus, or in close association with the cytoplasmic or nuclear membranes. These structures do not appear within the mitochondria. The rods composing this structure have a diameter of approximately 62 A. The chemical composition or functional role of these structures is unknown.

Cryobiological studies of yeast mitochondria

Summary Mitochondria from the yeast S. cerevisiae have been shown to be particularly suited for cryobiological studies, since they may be frozen and thawed without detectable injury to either structure or function. Mitochondria are frozen in the usual isolation medium containing mannitol and serum albumin, and no penetrating cryoprotective agent such as dimethyl sulfoxide or glycerol is required. Yeast cells have been stored at 4°C for at least 9 days, and isolated mitochondria for 30 days or more, in liquid N2 without appreciable change in mitochondrial Qo2, ADP:O, or adenosine diphosphate-linked respiratory control. Stored mitochondria retain normal morphology and capacity for adenine nucleotide translocation. Respiratory control has been preserved for as long as 53 days. Improved isolation methods together with these storage techniques have permitted study of intact yeast mitochondria with a facility virtually unattainable with mitochondria from other cell types. Mitochondria isolated from a yeast mutant with deficient aerobic energy metabolism exhibit increased lability during freezing and thawing. Both Qo2 and respiratory control are greatly reduced.