RNA Export and Electrokinetic Potential of Nuclei in Germinating Embryos of Cereal Crops

The changes in the contents of major components in the nuclei and nuclear membranes during germination of cereal crop embryos were studied. Treatment with RNase of intact nuclei from both dry and germinating embryos changed the electrokinetic potential (EKP) of the nuclear surface. The interrelations between an increased RNA export from isolated nuclei and increased EKP during germination were shown. The conclusion was drawn that the rate of RNA export from the nuclei affected substantially the EKP value, which opens new possibilities for studying physicochemical properties of the nuclear membrane in relation to the functional state of the genetic apparatus and the physiological state of the plant cell.     

Effects of hydrocortisone and insulin on the electrokinetic potential of hepatocyte nuclei

The electrokinetic potential of liver cell nuclei of rats after the in vivo separate and joint injection of hydrocortisone and insulin was determined. It was shown that the steroid induces a significant elevation of the value of electrokinetic potential while insulin does not change this parameter. An insignificant elevation of the potential by the joint action of the hormones was found, which apparently is indicative of the antagonism between hydrocortisone and insulin.     

Influence of amino acids on the activity of cellulolytic enzymes obtained from spent medium of Fusarium cultures and on the "Cellulase S" Novo.

Comparative studies on the influence of 4 amino acids (cysteine, cystine, methionine and alanine) on the activity of enzymatic preparation were carried out. The action of the amino acids was shown to depend on pH, incubation time and the temperature of incubation. Attempts were made to find a correlation between the amino acids introduced and redox potential (Eh), pH and the activity of the cellulolytic enzymes. It was found that the addition of some amino acids to the reaction mixtures at different pH values affected the value of Eh -- which enhanced the cellulase activity.     

Two different types of electrogenic amino acid action on pancreatic acinar cells

The electrogenic action of the basic amino acid, l-arginine, has been compared with the action of the neutral amino acids, l-alanine and glycine, in mouse pancreatic acinar cells. All three amino acids cause membrane depolarization, but while the reversal potential for the action of the neutral amino acids is close to the calculated value of the Na equilibrium potential (+30 m V) the reversal potential for the l-arginine effects is +7 m V. The neutral amino acids exhibit mutual inhibition, but l-arginine did not inhibit the l-alanine-or glycine-evoked depolarization nor did the neutral amino acids inhibit the action of l-arginine. While l-alanine markedly depressed acetylcholine-evoked depolarization, l-arginine had no such effect. It is concluded that there are at least two quite different types of electrogenic amino acid action in pancreatic acinar cells.     

Chemiluminescence in oxidation of luminol by chloramine derivatives of biogenic compounds

Chloramine derivatives of amino acids induce chemiluminescence of a luminol solution. The chemiluminescence is more prolonged than the emission of luminol produced by hypochlorite. Persistent chemiluminescence also appears under the action of hypochlorite on a mixture of luminol and amino acids. It is assumed that the chemiluminescence of luminol in suspensions of stimulated phagocytes may be associated with its oxidation by chloramines.     

Nuclear localization of gold labeled-hydrocortisone-bovine serum albumin conjugate injected intravenously into the hormone-target cells of rat.

We have suggested in a previous study using 2-nm colloidal gold labeled-testosterone-bovine serum albumin (testosterone-BSA-gold) that 2-nm gold labeled-steroid hormone-BSA conjugates would be a useful tool for analyzing the mechanism of steroid hormone action (39). In this study, we examined whether hydrocortisone-BSA conjugate (hydrocortisone-BSA) showed a similar distribution to radiolabeled hydrocortisone in vivo, by injecting 2-nm colloidal gold labeled-hydrocortisone-BSA (hydrocortisone-BSA-gold) into the rat tail vein. The hydrocortisone-BSA-gold with silver enhancement became visible as silver deposits under electron microscopy in the nuclei of hepatocytes and hepatic stellate cells but not in Kupffer cells in the liver, and in the thymocytes and thymic reticuloepithelial cells in the thymus of a rat killed 2 h postinjection. The percentage of nuclei showing deposits in the non-target cells, the epithelial cells of the seminal vesicle, was similar to the value in the seminal vesicle of a control rat injected with BSA labeled with 2-nm colloidal gold as reported previously. In the hepatocytes and thymocytes of a control rat not injected, the percentages of nuclei showing deposits were similar to those in the rat injected with testosterone-BSA-gold or BSA-gold as reported previously, but lower than those in the rat injected with hydrocortisone-BSA-gold. These results suggest that hydrocortisone-BSA-gold is useful for the morphological study of hydrocortisone target cells, and imply that BSA conjugated with hydrocortisone can enter the target cell nuclei of the rat. The present study further indicates that the fate of gold labeled-steroid hormone-BSA conjugates may be decided at the cell membrane level.     

Studies on the mechanism of induction of hypercholesterolemia in rabbits by high dietary levels of amino acids

Increasing the proportion of an amino acid mixture corresponding to casein in a low-fat, cholesterol-free, semipurified diet fed to rabbits causes a progressive increase in serum total and low density lipoprotein cholesterol, and the effect appears to be due primarily to the essential amino acids in the mixture. Our recent studies have also shown that the variations in serum cholesterol in response to different levels of the amino acid mixture are not associated with any changes in fecal excretion of cholesterol or bile acids. Further attempts to understand the mechanism of action of dietary amino acids on serum cholesterol levels have shown the following: (1) no correlation with levels of plasma amino acids, either in the fasting or postprandial state: (2) no correlation with serum levels of thyroid hormones: (3) no relationship to activity of hepatic or intestinal microsomal hydroxymethylglutaryl coenzyme A reductase: (4) no corresponding effects on the activities of cholesterol esterifying enzymes of intestinal mucosa: and (5) no correlation with the degree of esterification of cholesterol in very low or low density lipoproteins. Further studies are required to identify the specific amino acids responsible for the hypercholesterolemic effects and to determine the mechanisms involved.     

Control of cell-free protein synthesis by amino acids: effects on tRNA charging

In order to resolve the observation that addition of glutamine and glutamate appears to be of particular importance in enhancing the activity of a cell-free protein synthesis system derived from rat liver (Manchester and Tyobeka, 1980), we have measured the KM of the aminoacyl-tRNA synthetases towards amino acids and the extent of aminoacylation of tRNA under the conditions of our earlier experiments. During incubation of the cell-free system in the presence of an amino acid mixture the extent of acylation to tRNA of 15 amino acids studied showed no clear change from initial time values. When incubation took place in the absence of added amino acids, however, the levels of glutamate and glutamine bound to their appropriate tRNAs dropped more rapidly and to lower levels than for other amino acids except tryptophan. The pronounced drop for these two amino acids does not seem to result from an abnormally high KM value for the synthetases towards the respective amino acids, nor an abnormally low Vmax, but probably from the fact that the amounts of glutamyl and glutaminyl-tRNA in the cell-free system are comparatively low.     

Dependence of apparent viscosity on mycelial morphology of Streptomyces fradiae culture in various nitrogen sources

To examine what causes increased viscosity in culture broth in Streptomyces fradiae culture, various natural nitrogen sources were investigated. Extracellular protease activity increased with culture time and decomposed the natural nitrogen source into amino acids. In the case of gluten meal, after a culture time of 5 d, concentrations of glutamic acid and aspartic acid had increased to 600 and 200 mg/L, respectively, which were about 3- and 2-fold as high as levels in cultures under similar conditions using Pharmamedia. For various amino acids tested, the addition of glutamic acid or aspartic acid mixture to the culture medium raised the apparent viscosity to its highest demonstrated value, 260 mPa.s after 5 d of culture, which was 3-fold higher than without amino acids. Consumption of the decomposed glutamic acid and aspartic acid was dependent on the activities of glutamate dehydrogenase and aspartate aminotransferase, respectively. When ammonium ion was used as the nitrogen source, cell concentration reached 1.75 g/L measured as an intracellular nucleic acid concentration, which was about 2.3-fold higher than that with any other natural nitrogen source. However, apparent viscosity was only 75 mPa.s, a value one-third that of the amino acid mixture, and 70% of the pellets were bigger than 1.2 x 10(4) microm(2). In the case of gluten meal or the amino acid mixture, pellets bigger than 1.2 x 10(4) microm(2) comprised only 8%. This demonstrates that consumption of some amino acids affected the formation of filamentous morphology, which caused an increase in the apparent viscosity of the culture broth, and the apparent viscosity was not caused by the mycelial concentration but the mycelial morphology.     

Identification of heat-stable A-factor from Myxococcus xanthus.

The asg mutants of Myxococcus xanthus fail to produce a set of related substances called A-factor. A-factor is released into the medium and is required early in fruiting body development. Lacking A-factor, the asg mutants are defective in aggregation, sporulation, and expression of most genes whose products appear later than 1 h after development is induced by starvation. Previous work has shown that these defects are reversed when A-factor, released by developing wild-type cells, is added to asg mutant cells. Part of the material in conditioned medium with A-factor activity is heat stable and dialyzable. This low-molecular-weight A-factor consists of a mixture of amino acids and peptides. Fifteen single amino acids have A-factor activity, and 11 of these are found in conditioned medium. Mixtures of amino acids have a total activity approximately equal to the sum of the activities of their constituents. Conditioned medium also contains peptides with A-factor activity. Pure peptides have A-factor activity, and their specific activities are equal to or less than the sum of the activities of their constituent amino acids. There is no evidence for a specialized A-factor peptide in conditioned medium, one with a specific activity greater than the sum of its constituent amino acids. About half of the heat-stable A-factor activity in conditioned medium can be accounted for by free amino acids, and the remaining half can be accounted for by peptides. It is argued that heat-stable A-factor induces A-dependent gene expression not by the nutritional action of amino acids but through a chemosensory circuit.     

Studies on Amino Acid Contents of Processed Soybean

Effect of heating on the nutritive value of defatted soybean flour has been investigated by animal experiments. Loss due to heat degradation was evaluated in two ways. In the first method, the amino acids lost during overheating were supplemented by cystine and mixture of lysine, arginine, tryptophan, and serine at dietary levels of 1.6% nitrogen, and cystine and mixture of those amino acids plus histidine at dietary levels of 3.2% nitrogen. The other procedure adopted was the absorbent test used with amino acid mixtures based on the pattern of amino acids released by pancreatic hydrolysis of unheated, properly heated, and overheated defatted soybean flour at 6 and 120 hr hydrolysis.

At 1.6% dietary nitrogen level, the nutritive value of overheated soybean flour increased by supplementation with cystine and amino acid mixture, but at the 3.2% nitrogen level only cystine was effective. Supplementation of lost amino acids to overheated flour did not restore the nutritive value to that of the properly heated flour. Based on the amino acids released by pancreatic hydrolysis of unheated, properly heated, and overheated soybean flour after 6 and 120 hr reaction, amino acid mixtures were prepared and tested for their nutritive value. While the nutritive value of amino acid mixture prepared based on the pattern of amino acid liberated by 6 hr digestion of unheated, properly heated, and overheated flour did not show similar trend to that of 3 kinds of flour itself, the nutritive value of the amino acid mixture prepared after the data obtained by 120 hr digestion agreed well with the trend of unheated or heated soybean flour.

The nutritive value was also measured by the nitrogen balance of test animals.     

Aspartate and alanine aminotransferase activity in the tissues of adrenalectomized rabbits following administration of hydrocortisone and corticotropin

Aspartate and alanine aminotransferase (AsT, AlT) activities were studied in tissues of adrenalectomized rabbits which were treated with a single and multiple administrations of hydrocortisone (5 mg/kg) or a single administration of corticotropine (ACTH, 10 units/kg). It is shown that adrenalectomy decreases the AsT activity in homogenate of femoral muscle tissue and decreases the AlT activity in homogenate and supernatant of the liver, spleen and muscle tissue and in blood plasma. A single administration of hydrocortisone increases the AsT activity in supernatant of femoral muscle tissue and in blood plasma and increases AIT activity in the brain, liver, muscle and blood plasma. Parallel with that AsT and AlT activities are decreased in the spleen tissue. Multiple administration of hydrocortisone induces analogous changes in the AsT activity in the muscle and in the AlT activity in the liver, muscle and blood plasma. A single administration of ACTH induces an increase of the AsT activity in the muscle supernatant and in blood plasma. It also causes a rise of the AlT activity in the liver, muscle supernatant and blood plasma. The AlT activity is decreased in the brain supernatant. A question about stability of free amino acids metabolism (especially of alanine and aspartic acid) in the rabbit brain with changes in corticosteroid levels of organism is under discussion.     

Free amino acids in brain tissue and its subcellular structures in hyperthermia in rats

Free amino acids reserves in the brain tissue, nuclei and mitochondria were investigated in mongrel albino rats under conditions of hyperthermia (45 degrees C) of different duration--7,20 and 60 min. It was found that reserves of most free amino acids in the albino rat brain decreased under a short-term (7 min) hyperthermia and accumulated under more prolonged (20-60 min) one. The amount of amino acids in the brain mitochondria increased 7 min after the experiment start and decreased 20 min later, then (60 min later) the amount of most amino acids increased considerably. Opposite shifts were detected in the content of most amino acids in nuclei.     

The aerobic mineralization of organic compounds in the saline Lake Grevelingen (The Netherlands) (VI)

Summary During a one year period the uptake of aspartic acid and of a mixture of amino acids was determined using¹⁴C-labeled substrates as described by WRIGHT and HOBBIE (1966). By this technique the activity is analyzed of that part of the bacterial population which is able to utilize the added substrate. For comparison purposes the activity of the total heterotrophic bacterial population was determined by measurement of the oxygen consumption rate. From the oxygen consumption rate (mg O₂.l^–1.h^–1) the carbon mineralization rate (mg C.l^–1.h^–1) was calculated by applying a conversion factor of 0.29.Aspartic acid was respired for 80% and the amino acid mixture for 43%. From the maximum uptake rates, the potential yearly uptake of the substrate in question can be calculated. These data indicate the relative importance of the several subpopulations in the carbon mineralization process as a whole. The highest value of the potential yearly uptake was obtained for the amino acid mixture; the comparable value for the uptake of aspartic acid was slightly lower.The carbon mineralization rate as calculated from the oxygen uptake experiments was about 150–200 g C.m^–2.year^–1. The potential yearly uptake as determined with the¹⁴C-labeled amino acid mixture was only 2.8% of the amount of mineralized carbon, as calculated from the oxygen uptake experiments. This percentage is very low in view of the fact that 35–55% of the organic carbon of living phytoplankton and zooplankton consists of protein (HAGMEIER, 1961) and that the aerobic mineralization of amino acids is a very common property among the heterotrophic bacterial population (SEPERS, 1979). The value of the applied activity measurements was investigated in order to obtain information about the relation between the uptake process as measured with¹⁴C-labeled substrates and the activity of the bacterial population in situ. The results of this study have been published bij SEPERS and VAN ES (1979).     

Properties of RNA fractions from nuclei of brain cells which stimulate incorporation of amino acids by brain ribosomes

Abstract— The properties of RNA fractions from nuclei of brain cells which were capable of stimulating amino acid incorporation into proteins of an homologous ribosomal system were investigated. RNA was routinely prepared from crude nuclear preparations of rat brain by a method which involved treatment with sodium dodecyl sulphate and phenol at 65°. The capacity of this preparation to stimulate incorporation of radioactivity from a mixture of 15 l -[¹⁴C]amino acids was greatly enhanced by preliminary incubation of the ribosomal system from brain for 5–20 min. The response was markedly dependent upon the concentrations of ribosomes and of the pH 5 fraction. The optimal level of Mg²⁺ for basal incorporation of amino acids into protein was 8 mm ; however, incorporation in the presence of nuclear RNA was greater at higher concentrations of Mg²⁺. The response to nuclear RNA was also enhanced as the K⁺ concentration was increased from 25 to 100 mm . The stimulatory effect of nuclear RNA on incorporation of l -[¹²C]eucine was either unaltered or depressed by addition of a mixture of 19 l -[¹²C]amino acids each at concentrations, of 10^?8, 10^?2, or 10^?1 mm . Under appropriate conditions of incubation, basal rates of incorporation and rates of incorporation stimulated by nuclear RNA were linear for 30 min. The response was proportional to the concentration of nuclear RNA between 34 and 136 μg. RNA prepared from ribosomes of rat brain essentially failed to stimulate incorporation of amino acids over this range of concentrations. Fractionation of nuclear RNA by centrifugation in sucrose density gradients revealed that 75 per cent of the stimulatory activity was in the fraction which sedimented below 12 S and contained about 25 per cent of the total RNA. Most of the remaining activity was in the 18 S region. Less than 5 per cent of the RNA in the lightest fraction (< 12 S) exhibited amino acid-acceptor activity, The stimulatory action of nuclear RNA on incorporation of amino acids was readily destroyed by mild treatment with pancreatic ribonuclease, whereas amino acid-acceptor activity was relatively resistant to this treatment. The results suggest that the brain may contain low molecular weight RNA with properties of messenger RNA.     

Modification of rat liver RNA polymerase I after in vivo stimulation by hydrocortisone or methylisobutylxanthine

Following in vivo administration of hydrocortisone or methylisobutylxanthine to rats, higher levels (1.5- to 2.3-fold) of RNA polymerase I activity are present in liver nuclei and nucleoli of the treated animals as compared to control animals. The elevated specific activity is retained after purification of the enzyme under conditions where the enzyme is dependent on exogenous template for activity. The elevated polymerase activity in nuclei, nucleoli, and soluble enzyme can be destroyed by mild trypsin treatment which results in a rapid decay of the specific activity to the control level. Under these conditions, the control polymerase I activity is stable. The results indicate that in vivo stimulation by hydrocortisone or methylisobutylxanthine results in a conversion of the enzyme to a form that is catalytically more active but has an increased sensitivity to proteolysis.     

Evidence of amino acids in hydrolysates of compounds formed by ionizing radiations

It has been shown that the action of ionizing radiations on dilute, oxygen-free, aqueous solutions of acetonitrile and propionitrile leads to the formation of oligomers, which upon hydrolyses release amino acids. The presence of nine amino acids, the same as those found in irradiated aqueous cyanides, has been established. Those amino acids with asymmetric carbon atoms separated by GC method, appeared to consist of nearly equal amounts of D and L isomers. Glycine is the most abundant amino acid in hydrolysates of acetonitrile, while alanine appears in the samples of propionitrile. A comparison of all amino acids, identified in hydrolysates of various cyanides and nitriles, suggests that it is the cyano group, and a free-radical initiated mechanism, that is primarily involved in these radiation-chemical changes of potential interest to prebiotic chemistry.     

Evidence of amino acids in hydrolysates of compounds formed by ionizing radiations. II. Aqueous solutions of CH3CN and C2H5CN

It has been shown that the action of ionizing radiations on dilute, oxygen-free, aqueous solutions of acetonitrile and propionitrile leads to the formation of oligomers, which upon hydrolyses release amino acids. The presence of nine amino acids, the same as those found in irradiated aqueous cyanides, has been established. those amino acids with asymmetric carbon atoms separated by GC method, appeared to consist of nearly equal amounts of D and L isomers. Glycine is the most abundant amino acid in hydrolysates of acetonitrile, while alanine appears in the samples of propionitrile. A comparison of all amino acids, identified in hydrolysates of various cyanides and nitriles, suggests that it is the cyano group, and a free-radical initiated mechanism, that is primarily involved in these radiation-chemical changes of potential interest to prebiotic chemistry.     

Changes in rat liver chromatin after administration of a mixture of amino acids

It was shown that injections of an amino acid mixture essentially increase the number of nuclease-sensitive regions of chromatin and its active fraction (Mg2+-soluble fraction), while hydrocortisone increases the amount of the latter, which is less sensitive to the effect of DNAase II. Genome activation during nonhormonal induction after administration of the amino acid mixture or during hydrocortisone injection reflects in different ways on the parameters of melting of chromatin active fractions and on the relative content of its protein fractions.