ELECTRON MICROSCOPY OF CARTILAGE AND BONE MATRIX AT THE DISTAL EPIPHYSEAL LINE OF THE FEMUR IN THE NEWBORN INFANT

An examination of the fine structure of cartilage and bone matrix at the distal epiphyseal line of the femur of a newborn infant has revealed the following information. Cartilage matrix is composed of a network of widely spaced fibers without obvious periodic banding. Calcification is first seen about the level of the third chondrocyte capsule distal to the furthest penetration of the capillaries. It starts as a haphazard deposition of crystals which have no obvious relationship to the location of the fibers. The process of calcification is completed before ossification commences but the central zone of matrix remains only partly mineralized. Bone matrix is formed over a bar of calcified cartilage. Fibers, recognizable as collagen, are deposited in a loose network in a narrow zone between the osteoblasts and cartilage. These fibers are 2 to 5 times as wide as the fibers in epiphyseal cartilage. Calcification then begins in the osteoid, crystals being first laid down irregularly on or close to the fibers. As they increase in number, the crystals tend to line up along the fibers and eventually are arranged so that the periodicity of the underlying collagen is emphasized. In such an area the fibers are more tightly packed than when uncalcified. There is no change observed in the calcified cartilage at this level. The extracellular matrices of this epiphyseal cartilage and bone can be distinguished from one another in the electron microscope.     

NUCLEOLAR AND BIOCHEMICAL CHANGES DURING UNBALANCED GROWTH OF TETRAHYMENA PYRIFORMIS

Numerous nucleoli can be observed in the macronucleus of the logarithmically growing ciliated protozoan Tetrahymena pyriformis; at late log phase the nucleoli aggregate and fuse. In stationary phase this fusion process continues, leaving a very few large vacuolated nuclear fusion bodies in the nucleus. When these stationary phase cells are placed into fresh enriched proteose peptone medium, the large fusion bodies begin to disaggregate during the 2.5-hour lag phase before cell division is initiated. By 3 to 6 hours after inoculation the appearance of the nucleoli in many cells returns to what it was in logarithmic cells. In view of the possible role of nucleoli in ribosome synthesis, attempts were made to correlate the morphological changes to changes in RNA and protein metabolism. The beginning of an increased RNA synthesis was concomitant with the beginning of disaggregation of the large fusion bodies into nucleoli, which was noticed in some cells by 1 hour after the return to fresh enriched proteose peptone medium. Increased protein synthesis then followed the increased RNA synthesis by 1 hour. The supply of RNA precursors (essential pyrimidines) were removed from cultures which were grown on a chemically defined synthetic medium, in order to study the relation between nucleolar fusion and synthesis of RNA and protein. Pyrimidine deprivation drastically curtailed RNA and protein synthesis, but did not cause fusion of nucleoli. When pyrimidines were added back to this culture medium, RNA synthesis was immediately stimulated and again preceded an increased protein synthesis by 1 hour. These studies suggest the involvement of unfused nucleoli in RNA and protein synthesis and demonstrate the extreme plasticity of nucleoli with respect to changes in their environment.     

MACROMOLECULAR EVENTS LEADING TO CELL DIVISION IN TETRAHYMENA PYRIFORMIS AFTER REMOVAL AND REPLACEMENT OF REQUIRED PYRIMIDINES

Tetrahymena pyriformis were brought to a non-growing state by removal of pyrimidines from their growth medium. During pyrimidine deprivation cell number increased 3- to 4 fold, and this increase was accompanied by one or more complete cycles of macronuclear DNA replication. Autoradiographic studies show that endogenous protein and RNA were turning over throughout starvation and that RNA breakdown products were used to support the DNA synthesis that occurred during the early period of starvation. However, after 72 hours of starvation all DNA synthesis and cell division had ceased. Feulgen microspectrophotometry shows the macronuclei of these cells to have been stopped at a point prior to DNA replication (G1 stage). After pyrimidine replacement the incorporation of H³-uridine, H³-adenosine, and H³-leucine was measured by the autoradiographic grain counting method. The results indicate that RNA synthesis began to increase almost immediately, but that there was a lag of almost an hour before an increase in protein synthesis. In agreement with the autoradiographic data, chemical data also show that cellular content of RNA began to increase shortly after pyrimidine replacement but that cellular protein content did not increase until about one hour later. Pulse labeling of the cells with H³-thymidine at intervals after pyrimidine replacement shows that labeled macronuclei first began to appear at 150 minutes; that 98 per cent of the macronuclei were in DNA synthesis at 240 to 270 minutes; and that the percentage then began to decrease from 300 to 390 minutes, at which time only 25 per cent of the macronuclei were labeled. Cellular content of DNA did not increase for at least 135 minutes after pyrimidine replacement; however, just before the first cells divided (360 minutes) the DNA content had doubled. After pyrimidine replacement the cells first began to divide at 360 minutes, and 50 per cent had divided at 420 minutes; however, all cells had not divided until 573 minutes. This technique of chemical synchronization of cells in mass cultures makes feasible detailed biochemical analysis of events leading to nuclear DNA replication and cell division.