Striking nucleolar lesions occur in cultured cells after exposure to supranormal temperatures. These lesions appear at 42 degrees C and consist of a loss of the granular ribonucleoprotein (RNP) component and intranucleolar chromatin, and a disappearance of the nucleolar reticulum. The material remaining in the morphologically homogeneous nucleolus is a large amount of closely packed fibrillar RNP. The lesions remain identical as temperature increases to 45 degrees C. These alterations are reversible when the cells are returned to 37 degrees C and are associated with the reappearance of an exaggerated amount of intranucleolar chromatin and granular RNP. High-resolution radioautography indicates that after thermic shock nucleolar RNA synthesis is inhibited whereas extranucleolar sites are preserved: it also suggests that the granular RNP is reconverted to fibrillar RNP probably by simple unraveling. The results prove the existence of heat-sensitive cellular functions in the nucleolus which deal with the DNA-dependent RNA synthesis. The precise site of action is assumed to involve hydrogen bonds, resulting in configurational changes in nucleolar RNP and affecting the stability of the DNA molecule. The subsequent events in nucleolar RNA synthesis are discussed in light of the morphologic and biochemical effects of actinomycin D on the nucleolus.

Animals↗

PubMed1967

Ultrathin frozen sections. I. Methods and ultrastructural preservation.

A relatively simple method for obtaining ultrathin, frozen sections for electron microscopy has been developed. Tissues, cultured cells, and bacteria may be employed. They are fixed in 1.25-4% glutaraldehyde for 1-4 hr, are washed overnight in buffer at 3 degrees C, and are embedded in 20% thiolated gelatin or pure gelatin. Before sectioning they are partially dehydrated in 50% glycerol, frozen in liquid nitrogen on a modified tissue holder, and subsequently maintained at -70 degrees C with dry ice. Finally, they are sectioned very rapidly with glass knives on a slightly modified Porter-Blum MT-1 microtome in a commercial deep-freeze maintained at -35 degrees C and are floated in the trough of the knife on a 40% solution of dimethylsulfoxide (DMSO). The sections are picked up in plastic loops and transferred to distilled water at room temperature for thawing and removal of the DMSO, placed on grids coated with Formvar and carbon, air-dried, and stained with phosphotungstic acid, sodium silicotungstate, or a triple stain of osmium tetroxide, uranyl acetate, and lead. Large flat sections are obtained in which ultrastructural preservation is good. They are particularly useful for cytochemical studies.

Animals↗

PubMed1967

Ultrathin frozen sections. II. Demonstration of enzymic activity.

Endogenous enzyme activity can be readily and routinely demonstrated in ultrathin, frozen sections for electron microscopy. The procedure employed to obtain the best structural preservation as well as enzyme activity in thin sections involved fixation in glutaraldehyde, embedding in thiolated gelatin or pure gelatin, partial dehydration in glycerol, and sectioning in a cryostat at -35 degrees C with a slightly modified Porter-Blum microtome on which the tissue is maintained at -70 degrees C and the knife at -23 degrees C. Kidney cortex was used as test tissue, but a few other organs were occasionally used. Thin sections were floated on the surface of several incubation media routinely employed for enzyme cytochemistry. Positive, specific reactions were obtained for alkaline phosphatase in kidney brush border, for adenosine triphosphatase in brush border and in basal membranes of distal tubules, for acid phosphatase and esterase in lysosomes, and for NADH diaphorase in mitochondria. Mitochondrial ATPase was sporadically evident only in the distal tubule of the kidney. Localizations of enzyme activity reported by other technical approaches were confirmed and in some cases somewhat improved.

Acid Phosphatase↗

PubMed1967

Electron microscopy of adenovirus 12 replication. 1. Fine structural changes in the nucleus of infected KB cells.

The ultrastructure of KB cells infected with oncogenic adenovirus 12 was studied at various intervals from 4 to 72 hr after viral inoculation. At 12 hr after infection, the nucleus and the nucleolus became hypertrophic. At 16 hr, bundles of fibers digestable by proteolytic enzymes were seen in the nucleus; they are considered as the early viral antigens identified immunologically by others. Between 24 and 26 hr, four types of nuclear inclusions appeared. Their sequence of appearance and fine structure are described. On the basis of their sensitivity to proteolytic digestion in thin sections, and the results of immunoferritin studies made by others, some of these inclusions are believed to represent viral structural antigens. Throughout the cycle of viral replication, the nucleolus displayed prominent and constant changes in the form of focal condensations and loosening of the nucleolonema, followed by atrophy and fragmentation. It is suggested that the early nucleolar changes reflect an active participation of the nucleolus in the synthesis of adenovirus 12. A hitherto unknown striated structure with definite periodicity, which is easily digested by proteolytic enzymes, was found in the nuclei during the late stages of adenovirus 12 replication.

Adenoviridae↗

PubMed1967