Argininosuccinate synthetase and argininosuccinate lyase are localized around mitochondria: an immunocytochemical study.

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.

The neuropathology of intracerebroventricular t-butylhydroperoxide.

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.

Traversing filaments in desmosomal and hemidesmosomal attachments: freeze-fracture approaches toward their characterization.

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.

Retrieval of cryostat section for comparison of histochemistry and quantitative electron microscopy in a muscle fiber.

A method is presented that can be used to perform histochemical and morphometric analyses on the same muscle fiber. Freshly dissected fibers from medial gastrocnemius muscle of adult guinea pig were kept at a resting length and rapidly frozen. Serial frozen cross-sections were cut and reacted for myofibrillar adenosine triphosphatase and succinic dehydrogenase. The adjacent section, while still frozen, was immersed into 20 degrees C glutaraldehyde fixative to which EGTA was added to minimize artifactious contraction. The fixed section was processed for electron microscopy and the section rotated before thin sectioning to give longitudinal sections enabling study of sarcomeres. Ultrastructure was well-preserved despite slight disorganization of the contractile filaments and some vesiculation of the sarcoplasmic reticulum. The Z line width was measured and the mitochondrial volume fraction estimated by point counting morphometry from 89 fibers. The fibers with dark myofibrillar adenosine triphosphatase staining have Z widths of 547 +/- 165 A (n=69) and thoshosphatase staining have Z widths of 547 +/- 165 A (n=69) and those with light stain have 1023 +/- 113 A (n=20). The density of the succinic dehydrogenase reaction product in the fibers was divided into dark and light and the mitochondrial volume fractions were foud to be 4.3 +/- 2.1% (n=52) and 1.0 +/- 1.1% (n=37), respectively.

Stereological analysis of mammalian skeletal muscle. I. Soleus muscle of the adult guinea pig.

A quantitative analysis of the volumes, surface areas, and dimensions of the ultrastructural components in the soleus muscle fibers of the guinea pig was made by using point counting methods of stereology. Muscle fibers have structural orientation (anisotropy) and have spatial gradients of the structures within the fiber; therefore the standard stereological methods were modified where necessary. The entire analysis was repeated at two section orientations to test the modifications and identical results obtained from both. The volume of lipid droplets was 0.20 +/- 0.06% (mean +/- standard error, n = 5 animals) and the nuclei volume was 0.86 +/- 0.20% of the fiber volume. The total mitochondrial volume was 4.85 +/- 0.66% of the fiber volume with about one-third being found in an annulus within 1 microm of the sarcolemma. The mitochondrial volume in the remaining core of the fiber was 3.6 +/- 0.4%. The T system has a volume of 0.14 +/- 0.01% and a surface area of 0.064 +/- 0.005 microm(2)/microm(3) of the fiber volume. The surface area of the sarcolemma is 0.116 +/- 0.013 microm(2)/microm(3) which is twice the T system surface area. The volume of the entire sarcoplasmic reticulum is 3.52 +/- 0.33% and the surface area is 0.97 +/- 0.09 microm(2)/microm(3). The sarcoplasmic reticulum is composed of the terminal cisternae whose volume is 1.04 +/- 0.19% and surface area is 0.24 +/- 0.05 microm(2)/microm(3). The tubules of the sarcoplasmic reticulum in the I band and A band have volumes of 1.97 +/- 0.24% and 0.51 +/- 0.08%, and the surface areas of the I and A band reticulum are 0.56 +/- 0.07 microm(2)/microm(3) and 0.16 +/- 0.04 microm(2)/microm(3), respectively. The Z line width, myofibril and fiber diameters were measured.