Fluffy Layer Research Articles

Observations on iron uptake, iron metabolism, cytochrome c content, cytochrome a content and cytochrome c-oxidase activity in regenerating rat liver

1. Differential and density-gradient centrifugation were used to fractionate mitochondria and fluffy layer from normal and regenerating rat liver. The iron, cytochrome a and cytochrome c contents and cytochrome c-oxidase activity were studied as well as the uptake of (59)Fe into protein and cytochrome c. 2. A certain degree of heterogeneity was evident between the heavy-mitochondrial and light-mitochondrial fractions, and in their behaviour during liver regeneration. 3. The specific content of light-mitochondrial iron and cytochrome a was 1.3-1.4 times that of heavy mitochondria. Changes in cytochrome c-oxidase activity closely followed those of cytochrome a content during liver regeneration, but not for light mitochondria after 10 days. 4. Radioactive iron ((59)Fe) was most actively taken up by well-washed light mitochondria during early liver regeneration. After 22 days fluffy layer became preferentially labelled. This substantiates the view that fluffy layer partially represents broken-down mitochondria. 5. During early regeneration, light-mitochondrial fractions separated along a density gradient were about 3 times as radioactive, and showed distinct heterogeneity of (59)Fe-labelling, in contrast with near homogeneity for heavy mitochondria. 6. Immediately after partial hepatectomy fractions corresponding to density 1.155 were 5-10 times as radioactive as particles of greater density. The radioactivity decreased sharply after 6 days. 7. These particles of low density possessed higher NADH-cytochrome c-reductase (1.5-5-fold) and succinate-dehydrogenase (1.1-2-fold) activities than typical mitochondrial fractions. Their succinate-cytochrome c-reductase and cytochrome c-oxidase activities were slightly lower. 8. The results are discussed in relation to mitochondrial morphogenesis, and a possible route from submitochondrial particles is suggested.

SOME FEATURES OF MITOCHONDRIA AND FLUFFY LAYER IN REGENERATING RAT LIVER.

1. Mitochondria and fluffy layer were prepared from control and regenerating rat liver. Differential and density-gradient centrifugation were used to fractionate the preparations, which were examined for protein content, density and the activity of cytochrome c oxidase, succinate dehydrogenase, NAD-isocitrate dehydrogenase and NADP-isocitrate dehydrogenase. 2. During regeneration the mitochondrial protein content of the liver fell by 18% from the control value of 18.4mg. of protein/g. of liver (wet wt.) and by 3 weeks had risen to 130% of the control value. It then declined slowly. 3. The fluffy-layer protein content (4.7mg./g. of liver) varied inversely as the mitochondrial content and increased by 70% in the early stages (10 days) of liver regeneration. The results suggest that fluffy layer may partially represent both partly formed and broken-down mitochondria. 4. NAD- and NADP-isocitrate dehydrogenases differed in their behaviour during liver regeneration. 5. The succinate-dehydrogenase and NADP-isocitrate-dehydrogenase activity of fluffy layer was high and rose during the early stages of liver regeneration (1 week). Succinate dehydrogenase and cytochrome c oxidase were concentrated in the lighter fluffy-layer particles 10 days to 3 weeks after partial hepatectomy. The significance of this with respect to mitochondrial formation is discussed. 6. Mitochondrial fractions possessed a certain degree of heterogeneity in enzymic activity when separated according to size and density. The mean density of heavy mitochondria was 1.198, light mitochondria 1.193. Fluffy layer was nearly homogeneous in control liver, but during regeneration considerable heterogeneity became evident. The significance of the heterogeneity is discussed.

The Effects of Different Atmospheres and Various Supplements on the In vitro Survival and Growth of Ascaris suum Larvae

The in vitro cultivation of Ascaris larvae was carried out by supplementing either Eagle's medium or medium 199 with 20% calf or bovine serum. Growth was observed only when an initial concentration of 20,000 or more larvae/10 ml of medium were cultured at 37.5 C under partially anaerobic conditions. By this procedure a small percentage of the larvae reached sizes which made their isolation feasible. Continued development of the isolated larvae occurred only after the normal atmosphere was replaced by one of a nitrogen-5% CO2 mixture. Larvae were maintained for as long as 110 days and reached sizes up to 2,300 by 83 ,. In a previous paper (Cleeland and Laurence, 1962) a simple method for hatching large numbers of viable Ascaris larvae was described and the cultivation of these larvae in vitro in medium 199 (Morgan et al., 1950) supplemented with 20 to 30% bovine serum was reported. Survival for periods of 60 days at 37 C and 100 days at room temperature was observed along with growth of the larvae (up to 600 by 30 4) at the higher temperature when cultivation was carried out under partially anaerobic conditions. It has subsequently been found possible to increase both the time of survival and growth of the larvae. The present paper reports the results of these studies along with the effect of different atmospheric environments on their development and the initial attempts to define their nutritional requirements. MATERIALS AND METHODS Collection and preparation of infective eggs The collection and preparation of infective eggs were carried out as previously described (Cleeland and Laurence, 1962). However, most of the eggs used in the present study were obtained from the posterior two-thirds of the uteri of female worms, and a majority of these eggs never developed to the vermiform embryonic state. Consequently, to obtain enough larvae for study and eliminate the large amount of contaminating egg material, the following procedure was adopted. Hatching and preparation of the larval inoculum The egg suspensions containing approximately 10 to 20% infective eggs were washed several times with 0.01 M phosphate buffered saline, then resuspended in 20 ml of medium 199 to a concentration of 10,000 to 20,000 eggs/ml and transferred to a 125-ml Erlenmeyer flask. Hatching was carried out as reported earlier (Cleeland and Laurence, 1962) with a magnetic stirrer for a period of 24 hr at 22 to 25 C. Over 90% of the infective and a majority of the noninfective eggs were ruptured by this treatment, and most of the free larvae appeared to be undamaged and viable. The contents of two or three flasks were combined, centrifuged at 200 g for 10 min, and the supernatant fluid removed. The sediment was resuspended in 10 ml of fresh medium 199 and transferred to a 15-ml centrifuge tube. After centrifugation at 200 g for 10 min, the supernate was decanted leaving a sediment consisting of two distinct layers. The careful addition of 1 ml of 199 followed by gentle agitation of the tube caused most of the fluffy white upper layer to resuspend in the medium leaving the light brown lower layer intact. The resuspended material was decanted and the process repeated until only the brown layer remained. This layer, consisting mainly of live hatched larvae free of much of the eg material and all of the shell debris, was resuspended in 199 at room temperature until used (never more than 4 to 6 hr after hatching). Examination of the material in the white fluffy layer revealed that it consisted of damaged larvae, eggshells, egg debris, and from 20 to 40% of the viable larvae, depending on how carefully separation was carried out.

Effect of trypsin on liver microsomes.

The effect of trypsin on the morphology of the rat liver microsomal fraction isolated by differential centrifugation has been investigated. The microsomes were incubated at 37 degrees C and centrifuged thereafter under the conditions of their initial isolation. The trypsin-treated microsomes and the untreated controls were fixed in unbuffered osmium tetroxide and embedded first in gelose and then in methacrylate. In the trypsin-treated microsomes, there was a removal of the ribosomes from the rough vesicles. Parallel chemical determinations showed that the total nitrogen and total phosphorus of the pellet were lowered. Particles, densely stained with phosphotungstic acid (PTA) and homogeneous in appearance, were found within microsome smooth vesicles in a fluffy layer which collects on the top of the microsome pellet. The morphology of these PTA-stained particles remained unchanged after incubation with trypsin.

Isolation of soluble glucose-6-phosphatase from microsomes of bovine livers.

IN a recent report1, we described the isolation of soluble glucose-6-phosphatase from bovine liver by treating the microsomes with both sodium deoxycholate and digitonin. The treatment with sodium deoxycholate attacks the microsomes in such a manner that part of the inactive proteins is solubilized. The main part of active proteins is concentrated in the fluffy layer by centrifugation2. Using this method, we obtained better results in rendering digitonin soluble.

Separation of mitochondria into two morphologically and biochemically distinct types

The mitochondrial pellet obtained from rat liver by the fractionation procedure of Hogeboom et al. (5) consist of a densely packed lower layer containing mitochondria, overlaid by a loosely packed fluffy layer. By repeated washing and resedimentation the fluffy layer material has been separated from the mitochondria of the dense pellet and from the microsomes. This isolated fluffy layer material consists almost exclusively of particles which are distinctly different from the mitochondria of the dense layer and the microsomes. They are somewhat smaller than the mitochondria of the dense layer, but their similarity to these mitochondria in shape, osmotic behavior, and succinoxidase activity, suggests that they are mitochondrial in nature, and we have therefore called them “small mitochondria”. However, in contrast to the large mitochondria, which have an extremely low PNA to proteinnitrogen ratio (less than 10 per cent of that of the microsomes), the small mitochondria have a ratio more than 80 per cent of that of the microsomes. Their alkaline phosphatase activity is approximately twice that of the large mitochondria or microsomes, but their phospholipid content is intermediate. Large and small mitochondria, apparently similar to those of rat liver, have been obtained from a number of tissues.