Degradation of transplanted mitochondrial proteins by hepatocyte monolayers.

Reductively [3H]methylated rat mitochondria and mitochondrial-outer-membrane vesicles and mitochondrial-outer-membrane vesicles where monoamine oxidase is irreversibly labelled by [3H]pargyline have been transplanted into hepatocytes by poly(ethylene glycol)-mediated organelle or organelle-vesicle cell fusion. During subsequent culture of hepatocyte monolayers for 4-5 days, under conditions which mimic endogenous catabolic rates in vivo the transplanted organelle proteins retain their degradation characteristics observed in vivo (e.g. mitochondria: average t 1/2 72.5 h; monoamine oxidase: t1/2 55 h). In all cases protein degradation with first-order kinetics is only observed after an initial lag period (i.e. 24-30 h after fusion). Transplantation of fluorescein-conjugated organelles showed that the fluorescent material is rapidly internalized (average t1/2 1-6 h) and uniformly distributed in the cytoplasm. During a subsequent 18-24 h period (which corresponds to the lag period for intracellular destruction of transplanted mitochondrial material) the transplanted material is translocated to assume a perinuclear distribution. The destruction of transplanted mitochondrial proteins is compared with endogenous mitoribosomally synthesized proteins (average t1/2 52.5 h). Percoll fractionation of cell homogenates containing transplanted mitochondrial outer membranes where the enzyme monoamine oxidase is irreversibly labelled with [3H]pargyline shows a distribution of enzyme similar to lysosomal acid phosphatase. After transplantation of reductively methylated 3H-labelled mitochondrial-outer-membrane vesicles the cells were treated with leupeptin to alter lysosomal density. This treatment leads to the predominant association of acid phosphatase with dense structures, whereas the 3H-labelled transplanted material predominantly does not change density. Therefore transplanted mitochondrial-outer-membrane proteins are found in intracellular vesicular structures from which the proteins are donated for destruction, at least in part, by a lysosomal mechanism.     

Leg chaetotaxy and the classification of the Uropodina (Acari: Mesostigmata)

Details of the segmental chaetotaxy of the legs of 47 species of Uropodina are given. On the basis of the ontogenetic development of the chaetotaxy, the Uropodina may be divided into two groups which coincide with the concepts of the Lower (Polyaspidoidea) and Higher (Uropodoidea) Uropodina of certain authors. Chaetotactic criteria do not support the classification of the Polyaspidini and Trachyuropodini sensu Hirschmann and Z-Nicol within the Oplitinae Hirschmann & Z-Nicol, the Diarthrophallini within the Uroactiniinae Hirschmann & Z-Nicol or the genus Trachytes within the Uropodini.
A critical appraisal is given of the classification of the Uropodidae (based on "Gangmerk-male") by Hirschmann and Z-Nicol.     

Growth and lipolysis of rat adipose tissue: effect of age, body weight, and food intake

The purpose of the present work was to study age- and weight-controlled rats to determine which is the primary factor in reducing the lipolytic response of free fat cells and which has the greater effect on the ratio of fat cells to nonfat cells in adipose tissue. The method for estimating fat cell and nonfat cell numbers is based on the analysis of adipose tissue and fat cell DNA and lipid. In adequately fed rats, epididymal adipocyte hyperplasia is complete between 9 and 14 wk of age. Chronic underfeeding delays, but does not eliminate, normal fat cell hyperplasia and is accompanied by a net loss in the nonfat cell population. During 9-14 wk of age, rat epididymal adipose tissue enlarges mainly through adipocyte hypertrophy. Total fat cells from the epididymal adipose tissue of control rats represent only 20-23% of the total cell population. Chronic underfeeding increases the percentage of fat cells in the fat pad from 23 to 28%. Noradrenaline-stimulated lipolysis is proportional to fat cell numbers but is inhibited when fat cell lipid increases to over 80% of fat pad wet weight. Rat age is apparently not primarily responsible for the decreased noradrenaline-stimulated lipolysis in fat cells of 350-g rats in vitro.     

Complementary Functioning of Nitrogenase Components from a Blue-Green Alga and a Photosynthetic Bacterium

Nitrogenases from Anabaena cylindrica and Chloropseudomonas ethylicum were partially purified into two components. A. cylindrica fraction I protein complemented fraction II protein from C. ethylicum. However, the reciprocal cross between C. ethylicum fraction I and A. cylindrica fraction II was negative.     

STUDIES ON EPITHELIAL CELLS ISOLATED FROM GUINEA PIG SMALL INTESTINE

Sheets of mucosal epithelial cells were released from guinea pig small intestine after incubation with ethylenediaminetetraacetate. Cells in sheets retained their columnar shape for 24 hr at room temperature, and exclusion of nigrosine suggested they had intact plasma membranes. When sheets were disaggregated individual cells had normal morphology for at least 4 hr. During isolation 16% of the total protein and 24% of the total lactic dehydrogenase were lost from the cells, but subsequent enzyme leakage was low. Leakage increased with shaking, incubation at 37°C, or increasing the oxygen tension of the suspending medium, but was minimal when the Na⁺:K⁺ ratio in the medium was 8:1 and the osmolarity was high. Losses of particulate enzyme activities were negligible. Respiration was constant for up to 4 hr and was insensitive to calcium, bicarbonate, oxygen tension, and pH. It was inhibited by cyanide and iodoacetate and varied with the Na⁺:K⁺ ratio of the extracellular fluid and the structural integrity of the cells. All preparations concentrated potassium and excluded sodium, but lost this ability if ouabain was added or cells were broken. Potassium-42 uptake was also sensitive to temperature, ouabain, and structural integrity. The preparations are being used to study cell metabolism in the intestinal epithelium.     

Guinea-pig liver tryptophan pyrrolase. Absence of detectable apoenzyme activity and of hormonal induction by cortisol and possible regulation by tryptophan

1. When assayed in fresh homogenates, guinea-pig liver tryptophan pyrrolase exists only as holoenzyme. It does not respond to agents that activate or inhibit the rat liver enzyme in vitro. Only by aging (for 30min at 5 degrees C) does the guinea-pig enzyme develop a requirement for ascorbate. 2. The guinea-pig liver enzyme is activated by the administration of tryptophan but not cortisol, salicylate, ethanol or 5-aminolaevulinate. 3. The tryptophan enhancement of the guinea-pig liver pyrrolase activity is prevented by 0, 34 and 86% by pretreatment with actinomycin D, cycloheximide or allopurinol respectively. 4. The guinea-pig liver tryptophan pyrrolase is more sensitive to tryptophan administration than is the rat enzyme. On the other hand, the concentrations of tryptophan in sera and livers of guinea pigs are 45-52% less than those in rats. 5. It is suggested that tryptophan may regulate the activity of guinea-pig liver tryptophan pyrrolase by mobilizing a latent form of the enzyme whose primary function is the detoxication of its substrate.     

The enzymic and non-enzymic degradation of colneleic acid, an unsaturated fatty acid ether intermediate in the lipoxygenase pathway of linoleic acid oxidation in potato (Solanum tuberosum) tubers

Colneleic acid is an unsaturated ether fatty acid derived from linoleic acid via a lipoxygenase-mediated enzyme pathway. It is degraded (a) by an enzyme in potato tubers which is heat-labile and non-dialysable and (b) by a model system containing catalytic amounts of Fe(2+) ions. Both enzyme- and Fe(2+)-catalysed systems have similar properties with respect to pH optima (pH5.0-5.5), oxygen requirement (0.6-0.7 mol of O(2) consumed/mol of ether degraded), inhibitors and reaction products. An unstable product breaks down to C(8) and C(9) carbonyl fragments. Both systems are inhibited by low concentrations of antioxidants (e.g. 5mum-butylated hydroxytoluene) and some chelating agents (e.g. 5mum-diethyldithio-carbamate). The model system is strongly inhibited by metal ions, particularly Cu(2+) and Fe(3+), at 20mum. Hydrogen peroxide and haemoproteins do not substitute for the enzyme or Fe(2+) ions but the non-haem iron protein, ferredoxin, does catalyse the degradation.     

Interaction of the glucose tolerance factor (GTF) with insulin

Partially purified glucose tolerance factor (GTF) which had been extracted from Brewer's yeast was mixed with ¹²⁵I-insulin, and the solution was chromatographed on Sephadex G-50. Similarly, ¹²⁵I-insulin which had not been reacted with GTF was chromatographed. Insulin reacted with GTF produced a significantly greater effect on glucose uptake in epididymal tissue than that of native insulin. When GTF, exclusive of insulin, was chromatographed, the fraction which potentiated insulin activity had an elution volume greater than that of insulin. These results demonstrate that GTF binds to insulin. When insulin was reacted with acetic anhydride under conditions which acetylate the α and ε amino groups, GTF binding to insulin was inhibited. These results suggest that the α and ε amino groups of insulin may be involved in the binding of GTF to insulin.