Sidney Goldfischer

Peroxisomal and mitochondrial defects in the cerebro-hepato-renal syndrome.

The cerebro-hepato-renal syndrome is a rare familial malady with cerebral, renal, and skeletal abnormalities, severe hypotonia, cirrhosis, iron and lipid storage, and death within 6 months. Correlated electron microscopic, histochemical, and biochemical studies demonstrate defects in two oxidative organelles. Peroxisomes cannot be found in hepatocytes and renal proximal tubules. In hepatocytes and cortical astrocytes, mitochondria are distorted in their appearance and glycogen stores are increased. Oxygen consumnption of brain and liver mitochondrial preparations with succinate and with substrates reducing nicotinamide adenine dinucleotide is markedly diminished, but the consumption is normal with ascorbate and tetramethylphenylenediamine, which suggests a defect in electron transport prior to the cytochromes. Histochemical studies of mitochondrial oxidation point to a defect between the succinate dehydrogenase flavoprotein and coenzyme Q, possibly in the region of nonheme iron protein.

VISUALIZATION OF PEROXISOMES (MICROBODIES) AND MITOCHONDRIA WITH DIAMINOBENZIDINE

cacodviate buffer, till 7.4 (Sabatiiui, Beiusch atiti Barr,uett. J. Cell Biol. 17: 19. 1963), but formaldehyde 1115() ma ’ he used. J,ict,batio,i of 10-25 . frozeit sections or 25-40 i ,ionfrozensections (Sn ith and Farquhar. Sd. Ins/rum. News, 10: 13. 1965) is performed at 37#{176}C. Manganese iotis (0.001 .1!) stimulate peroxisome staining, i)ut manganese precipitates from solution at p11 9.0. At p11 8.0 or below, their stimulatory effect can be demonstrated readily. The p11 9 incubation medium consists of: 20 mg l)AB tetra-hlCl (Sigma Chemical Company, St. Louis, Mo.; 9.8 ml 0.05 M 2-amino-2-methyl-1,3-propandiol (Sigma) buffer, p11 10; 0.2 ml freshly Prepared 1% 11202. The p11 is adjusted to 9.0 if iiecessary and the precipitate that forms is filtered. Optimal staining of mitochondria occurs at a lower pH (6.0) and with one-twentieth the level of 11202. The p11 6 incubation medium consists of: 20 nig l)AB; 8.9 ml 0.05 M acetate buffer, p11 5 and adjustment to H 6; 1 ml 0.05 M manganese chloride; 0.1 nil freshly prepared 0.l 11202. The slight precipitate that forms is filtered. Incubation is performed at 37#{176}C. Fixation is generally 3-6 hr

THE LOCALIZATION OF PHOSPHATASE ACTIVITIES AT THE LEVEL OF ULTRASTRUCTURE

produ(-ts froni alkaline afl(l aci(l IItosl)hattLSe activities are detectallle in the electron microscope. Since then electron microscopy has demonstrated a number of reactiomi product localizations beyond the resolution of light microscopy. In several iuistaua-es it has removed the subjective element from localizations deduced from light microscopy. Although too little is known, particularly at levels of significance for electron microscopy, regar(liflg adsorption of reaction product or enzyme, and the mode of pre(-ipitation of metal 1)hOsllhates, significant intracellular localizations can now be described with coulsideral)le (-onfidenc(-, as imidicated in Text Fig. 1. In the early work, incubations for enzyme activities were performed on small blocks of tissime pre-fixe(1 iii either osmium tetroxide (briefly) or in formaldehyde-calcium; following incubation for enzyme activity the tissue was

THE CYTOCHEMICAL DEMONSTRATION OF LYSOSOMAL ARYL SULFATASE ACTIVITY BY LIGHT AND ELECTRON MICROSCOPY

mmmcd ismflu sized costec i’soolanmi nmo--coou’st ai oui tog umerve cells its thin Amaldite sectiomus sisowed a rati’sen wo-osk, chiflimse aosd soonmetim’s’ses granuumban flumcunescence as coummul000nedltco time very stnoo’sg green fluorescence so-en’s it’sparaflimo sect icon’s(8 t-o 10 , ) (Doohlstn#{246}n’s and Fouxe, op. cit.). This stromug fitmono-sceouce nnade it alnsuoost inu’sb)ossii)lotoo observe nuioson changes in amuse coostemmt cub the cell bodies prodtmced by certain piuamnm’sacobogic stut)stau’sces, e.g., MAO-inhihitoums. Tisios Amaldite sectiooms, ti’scro-bore, provicle os ossoore seomsitive i)asis for stmci’s expeninmen’st-s. Iou coosclumsiooum, tiuin so-ctium’ss of Araldite-enmbedded tissumes mmsoov booa voolunable complcnmueuut to the usual panafflo’s embedding, otTo-ring nmeaoss of overcoming (he gap between electron and flumoresco-nec nm’sicmooscopy, and providing a moore exact and sensitive basis for statistical aomabyses aumd for estin’sations of drung-inuduced changes of the ios(raneunonal an’siome levels.

NUCLEOSIDE DIPHOSPHATASE AND THIAMINE PYROPHOSPHATASE ACTIVITIES IN THE ENDOPLASMIC RETICULUM AND GOLGI APPARATUS

The effects of pH, fixatives and divalent ions on nucleoside diphosphatase (NDPase) and thiamine pyrophosphatase (TPPase) activities in the endoplasmic reticulum (ER) and Golgi apparatus (GA) were examined in adult and neonatal hepatocytes and other cell types in the rat. In liver cells TPPase and NDPase both have a similar localization in the rough ER, nuclear envelope and smooth ER but differ in their pH optima; TPPase is most active at pH 8, NDPase at pH 7. TPPase in the GA, unlike its counterpart in the ER, is most active at neutral pH. High levels of NDPase activity are present in the GA of neurons, epididymis and other cells, but not in hepatocytes. TPPase in the ER, but not the GA, is stimulated by the addition of adenosine triphosphate to the medium. These observations show that different conditions are required to demonstrate ER and GA diphosphatase activities. Whether separate enzymes or multiple configurations of a single protein are responsible for these activities cannot be determined by staining procedures.

THE DEMONSTRATION OF ACID HYDROLASE ACTIVITIES IN THE INCLUSION BODIES OF TYPE II ALVEOLAR CELLS AND OTHER LYSOSOMES IN THE RABBIT LUNG

Type II alveolar epithelial cells show high levels of acid phosphatase, aryl sulfatase B and β-glucuronidase activities. In formalin- and glutaraldehyde-fixed rabbit lung acid phosphatase and aryl sulfatase B activities were demonstrated in the cytoplasmic inclusion bodies, supporting their identification as lysosomes. Type II cells differ from alveolar macrophages in their levels of hydrolase activity and the fine structure of their active sites.

NUCLEOSIDEPHOSPHATASE ACTIVITIES OF CYTOMEMBRANES

Publisher Summary This chapter discusses the nucleosidephosphatase activities of cytomembranes. Light and electron microscopic studies are based on (1) nucleosidephosphatase activities in the plasma membranes of many cells, (2) nucleosidediphosphatase activity, of narrow substrate range, in the endoplasmic reticulum and nuclear membrane of some cells, (3) nucleosidediphosphatase activity, with wider substrate range, in the Golgi apparatus, and (4) acid phosphatase activity in the lysosomes. They illustrate the extent to which enzyme cytochemistry can be brought to the level of ultrastructure by incubation of simply prepared frozen sections of formalin-fixed tissues. The endoplasmic reticulum enzyme has been isolated from acetone powders of rat liver microsomes and beef adrenal, and its properties have been compared with those of the enzyme within frozen sections of fixed tissue. These observations lead to speculations regarding (1) the functional roles of the nucleosidephosphatases of different cytomembranes; (2) the intimate topographical relation that exists between Golgi lamellae and lysosomes; (3) the lysosomal nature of early secretory granules, and some mature ones; and (4) dynamic interrelations that may exist among the membranes of endoplasmic reticulum, Golgi apparatus, and lysosomes.

Cytochemistry of human catalase. The demonstration of hepatic and renal peroxisomes by a high temperature procedure.

The cytochemical demonstration of marker enzymes for subcellular organelles permits light microscopic analysis of their structure and function in normal and diseased tissues. Currently available staining procedures for the peroxidatic activity of catalase in peroxisomes of plant and animal cells yield weak and inconsistent light microscopic staining when applied to human tissues. We have developed a simple and sensitive high temperature procedure that clearly and reproducibly stains these abundant, but poorly understood, organelles in biopsy specimens of human liver and kidney. This method utilizes formaldehyde fixation, a modified diaminobenzidine (DAB) medium, incubation at 45 degrees C and postosmication for both light and electron microscopy.

CYTOPLASMIC COPPER AND ITS TOXIC EFFECTS: STUDIES IN INDIAN CHILDHOOD CIRRHOSIS

Morphological, histochemical, and chemical study of three necropsy specimens of liver in the terminal stage of Indian Childhood Cirrhosis revealed a strikingly high copper content. it is proposed that excess accumulation of copper in the cytoplasm of hepatocytes disturbs the microtubular system, causing hydropic swelling and the formation of Mallorys hyalin. Copper and copper-binding protein showed topographical association with Mallorys hyalin. Diffuse cytoplasmic staining and the lysosomal copper distribution also suggested that copper had a cytotoxic effect. The pattern of copper distribution in Indian Childhood Cirrhosis differs from that in Wilsons disease and in prolonged cholestasis with excessive hepatic copper deposition, indicating a different mechanism of the copper accumulation.

Arterial lysosomes and connective tissue in primate atherosclerosis and hypertension.

The cellular events that occur in the vessel wall consequent to changes in endothelial permeability result in the progression of vascular disease, particularly atherosclerosis. Female rhesus monkeys were fed an atherogenic diet or were made hypertensive for 6-8 months; and their vessels were then compared with vessels from control monkeys. Length-defined segments of coronary vessels, the thoracic aorta, and the abdominal aorta showed significant increases in total connective tissue in the atherosclerotic and hypertensive groups; pulmonary vessels did not. The diseased aortic segments had increased levels of two lysosomal enzymes, acid phosphatase and beta-N-acetylglucosaminidase; pulmonary vessels were not diseased and did not show these changes. Coronary vessels from the atherosclerotic and hypertensive groups did not show an increase in enzyme levels on biochemical measurements, but focal accumulations of lysosomes were identified by cytochemical techniques. In atherosclerotic lesions, a doubling of cholesterol and more than a tenfold increase in cholesterol ester were found. These connective tissue and lysosomal changes are early features of primate vascular disease and may result from the accumulation of excessive substrate (cholesterol ester) in the lysosomes of vascular smooth muscle cells.