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Argininosuccinate synthetase and argininosuccinate lyase are soluble cytoplasmic enzymes of the urea cycle. Previous biochemical studies using permeabilized hepatocytes showed that these enzymes are organized in situ, and function as if they are located next to the outer membrane of mitochondria. We have now confirmed and extended those observations in intact liver by means of immunocytochemistry at the electron microscope level. Morphometric analysis of the electron micrographs shows that argininosuccinate synthetase and argininosuccinate lyase are located in the immediate vicinity of the mitochondria, predominantly next to the cytoplasmic surface of the outer membrane. Some immuno-specific protein is also observed in the endoplasmic reticulum in the immediate vicinity of the mitochondria. These results support our previous biochemical findings, and additionally suggest that virtually all of the argininosuccinate synthetase and argininosuccinate lyase of the liver parenchymal cell are located just outside the mitochondria. © 1996 Wiley-Liss, Inc.

t-Butylhydroperoxide can be used as a model oxidative stress-inducing agent in the brain following intracerebroventricular administration. Mice were treated with saline, t-butanol, or t-butylhydroperoxide. t-Butanol is the major metabolite of t-butylhydroperoxide. t-Butylhydroperoxide had a number of effects, including that it damages dopaminergic, cholinergic, and GABAergic neurons as demonstrated immunohistochemically. Electron microscopic examination demonstrated that astrocytes, oligodendrocytes, endothelial cells, pericytes, and neurons are damaged by t-butylhydroperoxide. Dopamine and its metabolites were affected in a number of brain regions, as were serotonin and its metabolite. Choline acetyl transferase activity was decreased in the striatum. Edema was apparent as assessed by tissue protein levels. There was evidence of lipid peroxidation produced by t-butylhydroperoxide in the midbrain. t-Butylhydroperoxide is a neurotoxin that may be useful in understanding the unexpected ways the brain responds to oxidative stress.

Desmosomes and hemidesmosomes from larval newt epidermis were examined by freeze-fracture methods incorporating low osmolality fixation, short duration glycerination, complementary replica comparison, stereo imaging, and dark shadow printing. These procedures provide new evidence regarding the structure of "traversing" filaments as mediators of attachment between intermediate filaments and the cell membranes of desmosomes and hemidesmosomes. A detailed analysis of intramembranous particles and other structures in these attachments has also been possible. The relationship of this evidence to models of desmosomal structure suggested by other authors is discussed.

A quantitative analysis of the fast-twitch-glycolytic fibers of the adult guinea pig muscle has been made using the methods of stereology. This allows correlations with biochemical and physiological data to be made. The structural components involved in oxidative metabolism are found in small amounts, namely the volume fraction of blood vessel is 0.8 ± 0.3%; that of lipid droplets is 0.012 ± 0.006%; and that of mitochondria is 1.91 ± 0.18%. The entire sarcoplasmic reticulum is extensive as expected for a fast-twitch fiber, the volume being 4.59 ± 0.29% and the surface area being 132.4 ± 7.7 μm 2 /100 μm 3 of the fiber volume. The sarcolemma has a surface area of 9.7 ± 0.7 μm 2 /100 μm 3 which is more than half the T system surface area of 14.6 ± 0.6 μm 2 /100 μm 3 . The volume fraction of the T system is 0.27 ± 0.02%. The Z line width is narrow, being 610 ± 10 . Other measured parameters include nuclear volume, fiber, and myofibril diameters.

Stereological techniques are used to obtain quantitative information from electron micrographs from 300 fibers of skeletal muscle from the adult guinea pig. Ultrastructural parameters measured include Z line width, volume of mitochondria, volume and surface area of tsystem, terminal cisternae, and sarcoplasmic reticulum. Histograms, scattergrams, and statistical discriminant analysis are used to analyze the data. The whole fiber population can be separated with 90% success rate into the red and white vastus muscles and the soleus muscle, which are physiologically fast and slow, respectively. The soleus fibers have a Z line wider than 1100 A and smaller amounts of t-system and terminal cisternae than the vastus fibers. The mitochondrial volume of the vastus fibers shows a continuous distribution over a large range (0.1–15%), with the white vastus fibers at the lower end and the red vastus fibers at the higher end.

Freeze-fracture analysis of triadic junctions utilizing an abbreviated glycerination schedule reveals regularly arranged intramembranous subunits within junctional portions of sarcoplasmic reticulum terminal cisterns. The subunits appear as round to square 20-nm elevations of the junctional terminal cistern E-face; the complementary P-face is characterized by a concentration of small particles, the detailed arrangement of which is less obvious. The E-face subunits may contain a pore, and their spatial arrangement corresponds to the tetragonal array of dimples or feet previously described within the junctional gap. No corresponding subunits have been detected within T-tubular membranes. Stereo techniques and complementary replica analysis have been utilized for detailed study of the subunits as well as associated features of the fracture faces of both T-tubular and terminal cistern junctional and nonjunctional membranes. The new findings are discussed in relation to structural models which might explain the mechanisms of the triadic junction involved in excitation-contraction coupling.

The soleus muscle of adult guinea pigs is composed entirely of slow-twitch fibers which stain moderately for oxidative enzymes and lightly for myofibrillar ATPase. The homogeneity of fiber type allows a quantitative ultrastructural study to be made by the point counting stereological method of Weibel. Longitudinal sections of well-fixed muscle fibers were used to obtain the preliminary morphometric data. This analysis excludes the region within 1 µ of the sarcolemma where there may be a higher volume fraction of mitochondria. Transverse sections were taken to provide additional anatomical information.The mitochondria, which are elongated, are not uniformly distributed along the length of the fiber but tend to lie on either side of the Z disc (Figs. 1 and 4) and are sparse in the A band (Figs. 1 and 3).