Development of citrus fruits: Fruit development and enzymatic changes in juice vesicle tissue

Seasonal changes in enzyme activities and some components ofSatsuma mandarin and sweet lime were studied. Although the main acid in mature Satsuma mandarin fruit is citrate,malate was predominantly accumulated in the very early stageof fruit development. In sweet lime, malate was chiefly accumulatedthroughout fruit development. Juice vesicle tissue in Satsuma mandarin fruit developed infour distinctive stages. In the first stage, enzyme activitiesand the contents of protein and nucleic acid increased. Theactivity of phosphoenolpyruvate carboxylase increased most rapidly.Cell division was observed in the first half of this stage.In the second stage, acids accumulated remarkably but enzymeactivities and RNA content did not change. In the third, maturationstage, the content of RNA increased again. In the fourth stage,the contents of citrate and RNA decreased, whereas the activityof NAD-dependent isocitrate dehydrogenase increased. Compared with climacteric fruit, no remarkable increase in theactivity of NADP-dependent malic enzyme was observed in citrusfruit during maturation, while activities of citrate synthetaseand malate dehydrogenase increased fourfold. Respiratory activitydid not rise as prominently during that time. ¹ This paper is Contribution B-31, Fruit Tree Res. Stn. (Received February 7, 1977; )     

Starvation-induced impairment of metabolism in a freshwater catfish

Starvation induced changes in citrate synthase (CS), glucose-6-phosphate dehydrogenase (G6-PDH), lactate dehydrogenase (LDH), DNA, RNA, RNA/DNA ratio and protein were studied in the freshwater catfish Clarias batrachus. Starvation gradually decreased the activity of CS, G6-PDH and LDH in brain, liver and skeletal muscle of the freshwater catfish. The maximum reduction in these enzyme activities upto 35-45% was observed after 35 days of fasting. This shows substantial decline in aerobic and biosynthetic capacity during starvation period. DNA, RNA, RNA/DNA ratio and protein contents were also reduced from 40-67% which reflects reduction in an overall capacity of the protein synthesis. Starvation-induced macromolecular changes indicate impairment of metabolism in fish.     

The yctCBA operon of Yersinia ruckeri, involved in in vivo citrate uptake, is not required for virulence

A three-gene operon, named yctCBA (Yersinia citrate transporter), induced by citrate and repressed by glucose was identified from a previously selected in vivo-induced (ivi) clone in the fish pathogen Yersinia ruckeri. Interestingly, despite being an ivi clone, the drastic growth reduction of the yctC mutant in the presence of citrate, and the relatively high content of this compound in rainbow trout serum, the operon was not required for virulence.     

Correlation between cell cycle duration and RNA content.

The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA in Chinese hamster ovary (CHO) cells and in mitogen-stimulated human lymphocytes during their progression through the cell cycle. Green and red fluorescence of individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. CHO cells were synchronized by selective detachment at mitosis. Their rate of progression through G1 and subsequently through S phase correlated with the content of stainable RNA. The mean duration of the G1 phase was 5.2 hours for cells with high RNA content (highest 25 percentile population) and 8.1 hours for cells with low RNA (lowest 25 percentile). The duration of S phase was 5.9 and 7.5 hours for high- and low-RNA, 25 percentile subpopulations, respectively. Lymphocytes synchronized at the G1/S boundary by hydroxyurea or 5-fluorodeoxyuridine showed extremely high intercellular variation with respect to content of stainable RNA. After release from the block they traversed S phase at rates linearly proportional to the content of stainable RNA. The duration of S phase was five hours for cells with high RNA-, six to nine hours for cells with moderate RNA- and up to 27 hours for cells with minimal RNA-content. The data suggest that the rate of progression through the cell cycle of individual cells within a population may be correlated with the number of ribosomes per cell.     

Citric acid wastewater as electron donor for biological sulfate reduction

Citrate-containing wastewater is used as electron donor for sulfate reduction in a biological treatment plant for the removal of sulfate. The pathway of citrate conversion coupled to sulfate reduction and the microorganisms involved were investigated. Citrate was not a direct electron donor for the sulfate-reducing bacteria. Instead, citrate was fermented to mainly acetate and formate. These fermentation products served as electron donors for the sulfate-reducing bacteria. Sulfate reduction activities of the reactor biomass with acetate and formate were sufficiently high to explain the sulfate reduction rates that are required for the process. Two citrate-fermenting bacteria were isolated. Strain R210 was closest related to Trichococcus pasteurii (99.5% ribosomal RNA (rRNA) gene sequence similarity). The closest relative of strain S101 was Veillonella montepellierensis with an rRNA gene sequence similarity of 96.7%. Both strains had a complementary substrate range.     

Shifts in nuclear and cytoplasmic nucleic acid content in temperature-synchronized Tetrahymena pyriformis (HSM)

Nuclei were isolated by exposing temperature synchronized Tetrahymena pyriformis (HSM) to Triton-X-100. Cell division synchrony was induced with a repetitive 12-hour temperature cycle (9.5 hours at 13°, 2.5 hours at 29°). Increase in nucleic acid content was biphasic: primarily during the last two hours of the cold period well in advance of the synchronous burst of division and secondarily in the last hour of the warm period. Nuclear RNA content rises almost two hours ahead of cytoplasmic RNA which shows a maximum 0.5 hour before the onset of the warm period. The DNA content reaches a peak 30 minutes later. On the basis of these shifts there appears to be not net synthesis of nucleic acids during cell division. The changes in RNA/DNA of the isolated macronuclei and micronuclei suggest enhanced RNA turnover, loss to the cytoplasm and enhanced ribonuclease activity prior to cell division. Cytoplasmic RNA also appears to be subject to enzymic degradation.     

Scaling effects on metabolism of a teleost

Scaling effects on citrate synthase (CS), glucose-6-phosphate dehydrogenase (G6-PDH), RNA. RNA/DNA ratio and protein contents of brain, liver and skeletal muscle were studied in a teleost, Clarias batrachus. The activity of white skeletal muscle CS decreased significantly as a function of increasing body mass of the fish. It shows that the fulfilment of energy demand in white skeletal muscle is not dependent on aerobic metabolism. The activity of liver G6-PDH decreased with the increasing body mass showing reduction in NADPH generation for lipogenic activity. However, increase in G6-PDH activity showed enhancement in reductive synthesis in skeletal muscle of the larger-sized individuals. A positive scaling of RNA, RNA/DNA ratio and protein contents reflects changes in macromolecular turnover for ATP-supplying enzymes and proteins.     

Iron-shortage-induced increase in citric acid content and reduction of cytosolic aconitase activity in Citrus fruit vesicles and calli

Aconitase, which catalyses the conversion of citrate into isocitrate, requires Fe for its activity. The yeast and animal enzyme loses its enzymatic activity under Fe shortage and binds to RNA of genes involved in Fe homeostasis, altering their expression. Thus, the enzyme provides a regulatory link between organic acid metabolism and Fe cellular status. Roots and leaves of Fe-deficient plants show induction in organic acids, especially citrate. Although no RNA-binding activity has been so far demonstrated for the plant aconitase, whether alternations in enzyme activity by Fe could play a role in this induction remain unanswered. This question was investigated in lemon fruit [ Citrus limon (L.) Burm var Eureka ], characterized by the accumulation of citrate to about 0.3 M in the juice vesicles cells (pulp). Calli and isolated juice vesicles showed two- to three-fold induction in citrate level when subjected to Fe shortage. The mRNA level of aconitase exhibited no changes under reduced Fe concentrations. Analysis of aconitase isozymes demonstrated that out of two aconitase isozymes, typically detected in citrus fruit, only the cytosolic form displayed a reduced activity under low Fe concentrations. Our data support the notion of a limited Fe-availability-induced reduction in cytosolic aconitase, resulting in a slower rate of citrate breakdown and a concomitant increase in citrate levels.     

An investigation of the mechanism of activation of tryptophan by tryptophanyl-tRNA synthetase from beef pancreas

Exposure of dark-grown Euglena to white or red light, but not blue light, produced a twofold increase in the specific activity of citrate synthase. A 400-fold purification of mitochondrial citrate synthase (subunit Mr = 44000) was achieved from cells of Euglena gracilis by affinity chromatography on ATP-activated agarose. Antisera, raised against the homogeneously pure enzyme, were used to demonstrate that the increase in citrate synthase activity on exposure of dark-grown cells to light resulted from an increase in citrate synthase protein. Anti-(citrate synthase) was used to detect precursor citrate synthase resulting from the translation of total polyadenylated RNA from Euglena in a cell-free rabbit reticulocyte lysate system. Citrate synthase mRNA was found to be present in cells at all stages of regreening. However, extraction and translation of polyadenylated RNA from free polysomes isolated from darkgrown and regreening cells demonstrated that appreciable translation of citrate synthase mRNA was only occurring in regreening cells.     

Reduced nuclear triiodothyronine receptors in starvation-induced hypothyroidism.

Starvation of male rats during 48 hours causes a marked reduction of serum thyroxine, serum triiodothyronine, and liver nuclear triiodothyronine content. Liver nuclear receptors capacity for binding of triiodothyronine was reduced, in contrast to thyro-previc hypothyroidism in which binding capacity is normal. DNA-dependent RNA polymerase activities were reduced. In contrast to typical hypothyroidism, serum thyrotropin was low. This form of pituitary non-responsive hypothyroidism may represent a selective response to caloric and/or amino acid deprivation.     

Effect of estradiol on the RNA content and the activity of nucleolar RNA polymerase from rooster liver

Estradiol administration to roosters results in changes in the macromolecular composition of the liver. Besides a gradual increase in liver protein and a sudden enhancement of liver DNA after 24 hours, the most pronounced change occurs in the RNA content, viz. an increase up to 190% of controls starting 26 hours after estradiol administration.The activity of nucleolar RNA polymerase -solubilized and chromatographed on DEAE-Sephadex- is increased four-fold 26 hours after estradiol treatment. This increase was found to be due to an increased initiation frequency. Concurrently, the initiation characteristics of nucleolar RNA polymerase are changed.     

Some properties of ferric citrate relevant to the iron nutrition of plants

Ferric citrate, the form in which iron is transported in dicotyledonous plants, diffuses slowly through cotton cellulose dialysis membranes, used to serve as a model for plant cell walls. KCl at m M concentrations stimulates diffusion.Photoreduction of ferric citrate results in a rapid and nearly complete reduction of iron when the citrate concentration is low (50 M) as in the xylem sap of plants growing on non-calcareous soils. In 1 m M citrate, as in the xylem sap of plants that activate their Fe-efficiency reactions, fast reoxidation prevents the buildup of high ferrous levels until after citrate has been largely broken down by photodestruction.Photodestruction of citrate, catalyzed by iron, results in increase of pH in the solution and in the formation of a non-dialyzable form of iron, and thus can lead to deposition of inactive iron in leaves.     

Characterization of ribosomes from the myxomycete Physarum rigidum grown in pure culture

The plasmodial phase of the myxomycete Physarum rigidum, analyzed during the period of rapid growth, attained a ribonucleic acid (RNA) and protein content of 9.8 and 60.0%, respectively, on a dry weight basis. It possessed ribosomes of the 80S class which, especially in the absence of magnesium ions, partially dissociated to 60S and 40S subunit classes. Electron micrographs of ribosomes treated with uranyl acetate-lead citrate revealed a number of surface features. Nucleotide analyses of both ribosomal and total RNA disclosed that they were composed of 51.0 and 52.5% guanylic and cytidylic acids, respectively. Consistent with most reports on other organisms, guanylic acid was the most abundant nucleotide found in the various types of RNA and cytidylic acid was the least abundant. The S(0) (20,w) values of the total RNA classes, in 0.01 sodium acetate (pH 4.6) containing 0.10 m NaCl, were 5.2, 18.1, and 27.3 in S units. Changing the ionic environment of the RNA (0.017 molal potassium phosphate, pH 7.0, containing 0.01 m disodium ethylenediaminetetraacetate) resulted in a reduction of the S(0) (20,w) values to 4.2, 16.6, and 22.6 in S units, which is indicative of molecular conformational transitions. In general, the amino acid composition of the ribosomal proteins was similar to the data available on ribosomal proteins from other biological sources.     

Characterization of ribonucleic acids from the venom glands of Crotalus durissus terrificus (Ophidia, Reptilia) after manual extraction of the venom. Studies on template activity and base composition

RNA synthesis in the venom glands of Crotalus durissus terrificus was stimulated by the manual extraction of the venom (milking). RNA was extracted from venom glands activated by milking and fractionated by centrifugation through sucrose density gradients. Template activity for protein synthesis and base composition of the RNA fractions were studied. RNA fractions that sediment between 18S and 4S had the highest template activity. The base composition analysis indicated that the 28S and 18S rRNA have a C+G content of 65.4 and 58% respectively. The ;melting' temperature (T(m)) of DNA in 0.15m-NaCl-0.015m-trisodium citrate, pH7.0, was 85 degrees C, corresponding to a C+G content of 38%. The base ratio of the RNA fractions that showed a high template activity was intermediate between that of rRNA and homologous DNA. The possible role of these fractions in the synthesis of the two main toxins (crotoxin and crotamine) of the South American rattlesnake's venom is discussed.     

Biochemical responses of pea root tissue to cytokinin: enhanced rates of RNA synthesis

Rates of RNA synthesis were studied in cultured pea (Pisum sativum) root segments and cortical explants which require the hormone cytokinin for DNA replication and cell proliferation. Rate calculations were based on the specific radioactivity of the extracted RNA and the specific radioactivity of the extracted ATP pool after a pulse with ³H-adenosine. The kinetics of RNA synthesis was studied after 24 hours of culture with or without kinetin. We found that kinetin stimulated a 2- to 4-fold enhancement in the rate of RNA synthesis after 24 hours of culture as compared to controls. A similar order of magnitude of stimulation of RNA synthesis was found when RNA was isolated by cesium chloride centrifugation. Pulses during the first 24 hours indicate that kinetin stimulates the rate of RNA synthesis as early as 9 hours after treatment has begun. During the first 24 hours of culture, kinetin did not affect the specific radioactivity of the ATP pool. The ATP pool equilibrated slowly with the exogenous label (³H-adenosine) in the presence or absence of kinetin. After 3 days in culture, we found kinetin to cause an expansion of the extractable ATP pool and a corresponding reduction in the ATP pool specific radioactivity. We interpret these results to indicate a stimulation in the rate of RNA synthesis due to kinetin treatment prior to any other known response.     

Testosterone stimulation of mitochondrial aspartate aminotransferase in organ cultures of rat ventral prostate

Stimulation of mitochondrial aspartate aminotransferase (mAAT) activity by testosterone was determined in organ cultures of rat ventral prostate. The effect of testosterone on citrate accumulation in the culture medium was also determined. Testosterone stimulation of citrate accumulation and mAAT occurred in a dose dependent manner. Stimulation of mAAT activity occurred after a 1–3 h lag period and appeared to involve the synthesis of specific RNA since the response was inhibited by actinomycin D. Studies utilizing [³H]l-leucine indicated that unlike the total tissue, testosterone stimulated the incorporation of [³H]leucine into proteins of the mitochondrial fraction. The results suggested that mitochondrial proteins may be more sensitive to testosterone stimulation than cytosol proteins. The response was specific for mAAT since testosterone had no effect on mitochondrial malic dehydrogenase activity. The data suggested that testosterone may regulate prostate citrate content by the induction of mAAT in prostate mitochondria, which results in a source oxalacetic acid for citrate synthesis through transamination of aspartate by alpha ketoglutarate.     

Reduction of the nucleic acid content of single-cell protein concentrates

Methods for reducing the content of nucleic acid in protein concentrates from disintegrated yeast and microalgae were investigated. Protein concentrates were prepared by acid precipitation of extracted protein after cell wall separation. The influence of alkaline protein extraction on the content of RNA in isoelectrically precipitated protein concentrates was studied. It was found that when a strong decrease in the RNA content was obtained, this was followed by a decrease in the yield of protein concentrate. Protein concentrates were also prepared without cell wall separation by precipitation with different agents after cell disintegration. In the precipitates from microalgae, a RNA reduction was obtained. Precipitation of yeast, protein gave no essential reduction with the precipitants used. Precipitation of yeast protein by heating at an alkaline pH gave a protein concentrate with a low content of RNA. A slightly lower RNA content was obtained when the precipitation was performed in the presence of NaCl. The yield of amino acid nitrogen was 70–80% and the RNA content was 1–2%. A process with precipitation at alkaline pH for the production of microbial protein concentrates with a low content of nucleic acid is suggested.     

Beta-hydroxyaspartic acid in vitamin K-dependent plasma proteins from scorbutic and warfarin-treated guinea pigs

beta- Hydroxyaspartic acid is a rare amino acid, present in all vitamin K-dependent plasma proteins except prothrombin, and is formed by a post-translational hydroxylation of aspartic acid. We have now investigated whether this hydroxylation, like that of proline in collagen, is vitamin C-dependent. The vitamin K-dependent plasma proteins were isolated from normal and scorbutic guinea pig plasma by barium citrate adsorption and the beta- hydroxyaspartic acid content was determined. Compared with normal animals, scorbutic animals showed no significant reduction of beta- hydroxyaspartic acid content. In warfarin-treated animals there was a decreased content of both beta- hydroxyaspartic acid and gamma-carboxyglutamic acid in the barium citrate adsorbed fraction. It was concluded that the post-translational hydroxylation of aspartic acid is unlikely to be vitamin C-dependent.     

Effect of acidosis, alkalosis and monofluoroacetate administration on citrate and ATP content of rat renal medulla and papilla

1. --Renal distribution of citrate showed that there is an increase in citrate content from cortex to medulla and a decrease from medulla to papilla. Alkalosis produced an increase in citrate content and acidosis a decrease in renal citrate content, in each of the studied renal area. Monofluoroacetate produced no significant change in citrate content of medulla or papilla; it did not interfere with the acido-basic related changes in cortex citrate content, but its effect was additive. 2. --Renal distribution of ATP significantly decreased from cortex to medulla and from medulla to papilla. Acid or basic diet had no influence on intratissular ATP content. Fluoroacetate decreased renal ATP content.