Incorporation Of 3H-phenylalanine Research Articles

Studies on Hypoxia: XI. Long-Term Effects on the Epiphyseal Plate—A Histomeric and Radioautographic Study

Rats were exposed to hypoxia for one week. The mean thickness of epiphyseal plates form control rats was 430 micrometers (mum) which was reduced to 313 mum in hypoxic rats. Radioautographic incorporation of 3H-phenylalaine by connective tissue cells in hypoxic rats was reduced up to 38% in control rats.

GROWTH IN MONOLAYER CULTURE OF RAT PITUITARY CELLS WHICH SYNTHETIZE AND RELEASE ACTH

Cultures of rat pituitary gland cells were developed to study biosynthesis and release of ACTH. ACTH measurement was accomplished by radioimmunoassay. ACTH release was observed following stimulation with theophylline and cAMP in a dose-dependent manner. Biosynthesis was demonstrated by incorporation of 3-H-phenylalanine into the hormone, employing a double antibody technique.

Studies on hypoxia: VIII. Ultrastructural and Biochemical Effects of Prolonged Exposure on Rat Parotid Glands

The effect of chronic hypoxia upon the rat parotid gland is studied by electron microscopy and biochemistry. Male Sprague-Dawley rats are subjected to 88% N 2 and 12% O 2 at less than 2 psi pressure for 7 days. For electron microscopy, the tissues are perfusion-fixed, embedded and sectioned in a routine manner. In the biochemical studies, rats are injected with 3H-phenylalanine (2 μCi/g; s.i. ∼ 5 Ci/m M) 60 min before sacrifice. Fresh glands are then prepared for analysis of amylase (Cibachrome-amylase substrate method), DNA (diphenylamine reaction), and total protein (Lowry method). The radioactivity in the acid precipitable and soluble fractions is determined by liquid scintillation spectrometry. The control animals are pair-fed and handled identically except that they are maintained in an ambient atmosphere. The ultrastructure of the hypoxic cells is altered in several areas. The Golgi apparatus demonstrates a decrease in organization and contains fewer transport vesicles. The rough-surfaced endoplasmic reticulum is broken and presents many vesicular and concentric profiles. The mitochondria have undergone several changes including swelling, clumping of intramitochondrial matrix, and fragmentation of cristase. The nucleus demonstrates a fibrillar pattern and contains a reduced amount of heterochromatin. The biochemical results indicate that, compared with controls, the hypoxic cells contain only 55% of amylase and 84% of the DNA, and thus suggest a drastic reduction of exportable protein production and an increase in cellular size, respectively. On the contrary, the incorporation of 3H-phenylalanine demonstrates an increase in the amount of radioactivity in the acid precipitable fraction of the hypoxic cells. These results lead to the conclusion that hypoxic stress causes a suppression of exportable protein synthesis, but may induce other cytoplasmic protein production, reflecting the biochemical and morphologic adjustment of the cell to a recovery phase.

Metabolism of ethionine in ethionine-sensitive and ethionine-resistant cells of the enteric yeast Candida slooffii.

In a defined medium with added ethionine plus low methionine, phenylalanine, tryptophan, tyrosine, adenine, and additional methionine reversed inhibition of the enteric yeast Candida slooffii by ethionine. Isoleucine and 7-methylguanine restored half-maximal growth. Choline but not triethylcholine inhibited C. slooffii. 6-Mercaptopurine reversed ethionine inhibition and also synergistic inhibition by ethionine plus choline. Protection against ethionine by adenine plus aromatics was also evident with log-phase cells in the absence of methionine. Incorporation of ethionine-ethyl-1-(14)C by resting cells was partially inhibited by aromatic amino acids and methionine. Ethionine depressed incorporation of (3)H-phenylalanine but not of (3)H-adenine. Ethionine-resistant mutants were isolated which incorporated ethionine efficiently and degraded it to yet unidentified substances not including 5'-ethylthioadenosine. Ethionine-sensitive cells accumulated more S-adenosylethionine (SAE) than resistant mutants. Adenine was a good precursor of SAE. Radioactivity from ethionine-ethyl-1-(14)C was recovered from cell fractions of ethionine-sensitive cells with the following distribution: cold trichloroacetic acid-soluble > hot trichloroacetic acid-insoluble > lipids > deoxyribonucleic acid > ribonucleic acid. Total radioactivity recovered from ethionine-sensitive cells was twice as much as that from ethionine-resistant mutants.

Biosynthesis of 50S ribosomal proteins during the outgrowth of Bacillus subtilis spores.

The biosynthesis of 14 protein bands from 50S ribosomes was studied during the outgrowth of Bacillus subtilis W23 spores. The time at which the synthesis of each protein band begins and the patterns of synthesis that each displays were determined by comparing the incorporation of (3)H-phenylalanine during five intervals throughout the first hour of outgrowth with the incorporation of (14)C-phenylalanine between 145 and 150 min. The majority of the proteins in the 14 bands analyzed were synthesized as early as 5 to 10 min. The bands could be grouped on the basis of their labeling patterns as well as the time required for them to achieve a high rate of synthesis. Three bands did not achieve a high rate of synthesis until after 30 min of outgrowth, and at least one of the proteins from these bands appeared to be located on the exterior of the 50S subunit. The significance of the proteins from these three bands is discussed in terms of the late formation of new, mature 50S subunits during outgrowth.

Effect of antihistamines on ribonucleic acid synthesis in the uterus of the ovariectomized rat.

To obtain information on the role of histamine in the action of estrogen on the uterus, the in vitro incorporation of 3H-uridine into ribonucleic acid (RNA) of uterine horns was measured following treatment with histamine and/or antihistamines. Compounds were either administered to uterine horns intraluminally immediately following cervical dislocation of the rat or were added to incubation media. Benadryl treatment caused a dose dependent decrease in incorporation of the RNA precursor when compared with controls treated with vehicle. A similar response was observed using adrenal bisects and segments of small intestine in in vitro studies. Compounds from 3 classes of antihistamines and others, structurally related to Benadryl but having low potencies as antihistamines, also reduced the incorporation of 3H-uridine into RNA. This effect was also observed in in vivo studies using Benadryl and its analogues. There was a significant decrease in the incorporation of l4C-acetate into protein of the castrate rat uterus under the influence of 0.70 mg Benadryl; however, there was no significant change in the incorporation into total lipids, or of 3H-phenylalanine incorporation into protein using either 0.70 or 0.35 mg antihistamine. Oxygen uptake by the tissue was severely impaired under the influence of Benadryl at and above 1.00 mg dose level. Using a turbidometric method it was shown that all the antihistamines tested formed insoluble complexes with RNA prepared from rat liver. This study indicates the need for caution in the interpretation of results obtained from any investigation in which antihistamine effects are used as indicators of an involvement of histamine in growth processes. (Endocrinology85: 778, 1969)

Early changes in kangaroo lymphocytes stimulated with phytohaemagglutinin

Kangaroo lymphocytes exposed to PHA show a rapid increase in the rate of synthesis of RNA; labelled RNA is at first confined to the nucleus, but soon appears in the cytoplasm. This is followed by increases in the rate of incorporation of 3H-phenylalanine, in nuclear and cytoplasmic volume, and in nuclear dry mass. DNA synthesis in cultures of kangaroo lymphocytes exposed to PHA occurs 12 h earlier than in similar cultures of human lymphocytes, and it is suggested that this is due to the earlier onset of protein synthesis in the marsupial lymphocytes.

Further confirmation of “division protein” fraction in Tetrahymena pyriformis

1. 1. In logarithmically growing Tetrahymena cells (at 26 °C), when the temperature was raised to 34 °C, 3H-phenylalanine incorporation in water-soluble proteins chromatographed on DEAE-cellulose decreased. In particular, the activity of the fraction designated as “peak 7” of water-soluble proteins was affected most seriously. 2. 2. On the other hand, around the so-called critical point in the recovery period, increases of 3H-methionine incorporation were found in peaks 4 a, 4 b, 6 b and 7 a and most strikingly in 7 a (the first half of peak 7). 3. 3. On these two evidences, our previous supposition that division protein is present in peak 7 is further consolidated.