A characterization of ribonucleic acid in the myxomycete Physarum polycephalum

This report deals with the quantitative extraction of total nucleic acid (TNA) containing undegraded RNA from the slime mold Physarum polycephalum. With the use of a three-step phenol extraction technique, approx. 95 % of the nucleic acid optical density and 90 % of the ³H-uridine incorporated radioactivity were routinely recovered in the extracts. With the use of this technique it was shown that (1) the TNA mg dry wt of the mold did not change throughout the mitotic cycle, even though the dry wt doubled; this indicates a continual net synthesis of nucleic acid throughout the cycle; (2) the relative proportions of the various nucleic acid components did not change significantly during the cycle and were found to be DNA, 6 %; rRNA, 82 %; and sRNA, 12 %; (3) RNA molecules with mol wts of 4.1 m and 1.9 m, which exhibit properties of rRNA precursors were found in plasmodia labeled for 20 min with ³H-uridine. Furthermore, there appears to be an RNA fraction, found only in nucleic acid preparations presumably enriched in nuclear RNA components, which is heat-labile, does not enter 2.6 % acrylamide gels during 4 h of electrophoresis, and has a uridine/methyl ratio different from the presumed rRNA precursors and mature rRNA.     

Differences in incorporation of nucleic acid bases and nucleosides by various Mycoplasma and Acholeplasma species.

Eight species representative of the serological diversity of the Mycoplasmatales were tested for their ability to incorporate radiolabeled nucleic acid precursors into acid-insoluble material. Cultures in complex growth medium were centrifuged and resuspended in minimal essential medium (Eagle). For Acholeplasma laidlawii, labeling occurred mainly during the first 4 h of incubation, with substrate saturation at 20 micron. All organisms tested incorporated uracil, adenine, and guanine; none incorporated cytosine. Thymine was incorporated only by bovine group 7, Mycoplasma putrefaciens, and Mycoplasma pneumoniae (strain 3546), but deoxynucleosides enhanced thymine incorporation in A. laidlawii, Mycoplasma gallisepticum, M. pneumoniae (strain AP-164), and Mycoplasma hyorhinis. Nucleoside incorporation (adenosine, guanosine, uridine, cytidine, and thymidine) was not observed for the arginine-utilizing species, Mycoplasma hominis and Mycoplasma arginini, whereas all other organisms tested incorporated nucleosides. The incorporation pattern provides additional metabolic evidence to support the biochemical and antigenic diversity of these organisms. The recognition of differences in incorporation of nucleic acid precursors is important not only to the specific labeling of these organisms, but also to the study of metabolism and transport.     

Efficient Production of Lactic Acid from Sweet Sorghum Juice by a Newly Isolated Lactobacillus salivarius CGMCC 7.75

Sweet sorghum juice was a cheap and renewable resource, and also a potential carbon source for the fermentation production of lactic acid (LA) by a lactic acid bacterium. One newly isolated strain Lactobacillus salivarius CGMCC 7.75 showed the ability to produce the highest yield and optical purity of LA from sweet sorghum juice. Studies of feeding different concentrations of sweet sorghum juice and nitrogen source suggested the optimal concentrations of fermentation were 325 ml l⁻¹ and 20 g l⁻¹, respectively. This combination produced 142.49 g l⁻¹ LA with a productivity level of 0.90 g of LA per gram of sugars consumed. The results indicated the high LA concentration achieved using L. salivarius CGMCC 7.75 not only gives cheap industrial product, but also broaden the application of sweet sorghum.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-013-0377-0) contains supplementary material, which is available to authorized users.     

Stability of the Nucleic Acids of L Cells After Infection with the Meningopneumonitis Agent

The stability of host nucleic acids in L cells infected with Chlamydia psittaci (strain meningopneumonitis) was studied. The L cells were prelabeled with either (32)P-orthophosphate, (3)H-uridine, or (3)H-thymidine. After infection, the redistribution of each label among the different fractions of host and parasite was quantitatively determined and compared. There were no signs of accelerated degradation of host nucleic acid as the consequence of meningopneumonitis infection. Comparison of the specific activities of the meningopneumonitis nucleic acids with that of the acid-soluble fraction of host cell cytoplasm suggested that the major source of precursors for parasite nucleic acid synthesis was the common cytoplasmic pool of the infected host cell.     

The incorporation of radio-labelled nucleic acid precursors by Theileria parva in bovine blood and salivary glands of Rhipicephalus appendiculatus ticks

Irvin A.D., Boarer C.D.H., Kurtti T.J. and Ocama J.G.R. 1981. The incorporation of radio-labelled nucleic acid precursors by Theileria parva in bovine blood and salivary glands of Rhipicephalus appendiculatus ticks. International Journal for Parasitology11:451–456. The uptake of radio-labelled nucleic acid precursors by blood and tick salivary gland forms of Theileria pana was studied. Piroplasms took up tritiated purines, particularly hypoxanthine, but not pyrimidines. Similar uptake was recorded by T. parva, both in tick saliva and in salivary glands maintained in vitro. Intermediate parasite stages were those most readily labelled in glands; this reflected active nucleic acid synthesis associated with rapid parasite division. Radio-labelling of T. parva in tick salivary glands could be of value in procedures used for concentrating and purifying theilerial sporozoites.     

In vivo assessment of precursor induced prostaglandin release within the rat gastric lumen

Gastroprotection associated with the intragastric administration of prostaglandin (PG) precursor fatty acids such as linoleic (LA), gamma-linolenic (GLA), and arachidionic acid (AA) has been reported to be mediated via their conversion to PGs. This report examines the relationship between gastroprotection and the extent/rate of PG-release in rats intragastrically administered PG biosynthetic precursors: LA, AA, dihomo-gamma-linolenic acid (DHGL) or oleic acid (OA, a nonprecursor fatty acid). At various times following intragastic administration of a fatty acid, gastric fluid was collected, extracted, chromatographed, and assayed for PGE₁ or PGE₂ by specific radioimmunoassay. AA and DHGL dose dependently elevated gastric PGE₂ and PGE₁ levels, respectively. Maximal PGE elevation, 200–400 ng/stomach, was over 400-fold above basal values, and observed within 5–10 minutes of administration. Conversely, OA and LA elicited only a minor (2–10 fold) stimulation of PGE release. In contrast to effects on PG release, all four fatty acids protected the gastric mucosa against macroscopic damage induced by ethanol. The apparent rank order of potency was AA > DHGL = LA > OA (the difference in potency between DHGL or LA and OA was not significant). Since LA and OA (a nonprecursor) only marginally elevated lumenal PGs relative to DHGL or AA, yet were equally efficacious in the gastroprotection assay, it is likely the other fatty acid-related mechanisms play an important role in protecting the stomach against ethanol-induced injury.     

Effects of human peripheral blood monocytes,monocyte-derived macrophages,and spleen mononuclear phagocytes on Toxoplasma gondii

In vitro effects of human peripheral blood monocytes, peripheral blood monocyte-derived macrophages, and spleen mononuclear phagocytes on Toxoplasma gondii were studied. In almost all instances, over 80% of human monocytes and monocyte-derived macrophages infected with Toxoplasma in vitro destroyed the organism. Degeneration of intracellular Toxoplasma was not due to decreased viability of organisms in the challenge inoculum. Human monocytes did not elaborate into the culture medium substances which altered the capacity of Toxoplasma to survive and replicate within mouse macrophages. The early reduction in intracellular Toxoplasma was not affected by inhibitors of various intracellular processes or by diseases associated with altered cellular immunity (sarcoidosis, infectious mononucleosis, or lymphoma.) The Toxoplasma that remained after 6 hr within human monocytes and macrophages multiplied. This multiplication was observed both microscopically and in a radioassay which detects uptake of [³H]uracil or [³H]deoxyuridine into nucleic acids of intracellular Toxoplasma. Intracellular Toxoplasma in monocytes cultured with poly(I:C) or in monocyte-derived macrophages cultured with lymphokines showed decreased uptake of radiolabeled precursors into nucleic acids of intracellular Toxoplasma. Treatment of monocytes with endotoxin did not alter nucleic acid synthesis of surviving intracellular Toxoplasma. These results suggest that human mononuclear phagocytes in peripheral blood and in tissue (spleen) have the capacity to eliminate a large percentage of the Toxoplasma that they ingest or that invade them. The inhibition of nucleic acid synthesis of remaining Toxoplasma by exposure of monocyte-derived macrophages to lymphokines suggests that lymphocyte products may be important for elimination of the Toxoplasma that remain and multiply within a small proportion of mononuclear phagocytes.     

Free fatty acids from the pasture grass Brachiaria humidicola and one of their methyl esters as inhibitors of nitrification

The tropical pasture grass, Brachiaria humidicola (Rendle) Schweick, produces nitrification inhibitory compounds (termed biological nitrification inhibitors or BNIs) in its shoot and root tissues and releases BNIs from its roots. In the present study, two BNI compounds were isolated and identified from the shoot tissue of B. humidicola using activity-guided fractionation. The recombinant Nitrosomonas europaea containing luxAB genes derived from the bioluminescent marine gram-negative bacterium Vibrio harveyi, were used to determine BNI activity. The BNI compounds in the shoot tissue were identified as linoleic acid (LA) and linolenic acid (LN) using authentic-chemicals obtained from ©Sigma (ED₈₀ 16.0 μg ml^?1 for both LA and LN) for verification. None of the other tested free fatty acids namely stearic acid, oleic acid, arachidonic acid, and cis-vaccenic acid showed any inhibitory effect on nitrification. Among the fatty acid methyl esters (FAME) evaluated [methyl oleate, methyl linoleate (LA-ME) and methyl linoleneate (LN-ME)], only LA-ME showed an inhibitory effect (ED₈₀ 8.0 μg ml^?1). The inhibitory effect of LA, LN and LA-ME in the soil was stable for 120 days at 20°C. Soil treated with LA, LN and LA-ME showed a very low accumulation of NO₃ ^? and the maintenance of soil inorganic N in the NH₄ ⁺ form. The inhibitory effect of LA-ME on soil nitrification was greater than that of LA or LN. In addition to BNI activity, both LA and LA-ME showed a suppressive effect on urea hydrolysis in soil. Both LA and LN blocked the AMO (ammonia monooxygenase) and HAO (hydroxylamino oxidoreductase) enzymatic pathways in Nitrosomonas. Since LA and LN can be produced from vegetable oils such as soybean, flax or sunflower, they have the potential for use as nitrification inhibitors in production agriculture.     

Lipoic Acid Does Not Affect The Growth of Mycoplasma hominis Cells In Vitro

Mycoplasma hominis is associated with various infections, for which the treatment can be complex. Lipoic acid (LA) plays a role as a cofactor in eukaryotes, most Bacteria, and some Archea. Research of recent years has increasingly pointed to the therapeutic properties of exogenously supplemented LA. The present study was conducted on 40 strains of M. hominis cultured with the following LA concentrations: 1,200 μg/ml, 120 μg/ml, and 12 μg/ml. The bacterial colonies of each strain were counted and expressed as the number of colony-forming units/ml (CFU). The number of CFU in M. hominis strains obtained in the presence of LA was compared with the number of CFU in the strains grown in the media without LA. The obtained results indicated that the presence of LA in the medium did not affect the growth of M. hominis. The investigation of the influence of LA on the growth and survival of microbial cells not only allows for obtaining an answer to the question of whether LA has antimicrobial activity and, therefore, can be used as a drug supporting the treatment of patients infected with a given pathogenic microorganism. Such studies are also crucial for a better understanding of LA metabolism in the microbial cells, which is also important for the search for new antimicrobial drugs. This research is, therefore, an introduction to such further studies.     

Interactive effects of maternal and weaning high linoleic acid intake on hepatic lipid metabolism,oxylipins profile and hepatic steatosis in offspring

Non-alcoholic fatty liver disease (NAFLD) has been described as a hepatic manifestation of the metabolic syndrome. When several studies correlated maternal linoleic acid (LA) intake with the development of obesity, only few links have been made between n-6 fatty acid (FA) and NAFLD. Herein, we investigated the influence of both maternal and weaning high LA intake on lipid metabolism and susceptibility to develop later metabolic diseases in offspring. Pregnant rats were fed a control-diet (2% LA) or a LA-rich diet (12% LA) during gestation and lactation. At weaning, offspring was assigned to one of the two diets, i.e., either maintained on the same maternal diet or fed the other diet for 6 months. Physiological, biochemical parameters and hepatic FA metabolism were analyzed. We demonstrated that the interaction between the maternal and weaning LA intake altered metabolism in offspring and could lead to hepatic steatosis. This phenotype was associated with altered hepatic FA content and lipid metabolism. Interaction between maternal and weaning LA intake led to a specific pattern of n-6 and n-3 oxylipins that could participate to the development of hepatic steatosis in offspring. Our findings highlight the significant interaction between maternal and weaning high LA intake to predispose offspring to later metabolic disease and support the predictive adaptive response hypothesis.     

The effects of levulinic Acid and 4,6-dioxoheptanoic Acid on the metabolism of etiolated and greening barley leaves

Application of levulinic acid (LA), a competitive inhibitor of δ-aminolevulinic acid (ALA) dehydratase, to greening plant tissues causes ALA to accumulate at the expense of chlorophyll. 4,6-Dioxoheptanoic acid (DA), which has been reported to be an effective inhibitor of this enzyme in animal systems, has a similar but more powerful effect on ALA and chlorophyll metabolism in greening leaves of Hordeum vulgare L. var. Larker. Both LA and DA also inhibit the uptake of [¹⁴C]amino acids into etiolated and greening barley leaves and reduce their incorporation into protein. Treatment of etiolated and greening leaves with these compounds results in the inhibition of ¹⁴CO₂ evolution from labeled precursors, including amino and organic acids. Inhibition of ¹⁴CO₂ evolution by these compounds is more effective in greening leaves than in etiolated leaves when [4-¹⁴C]ALA or [1-¹⁴C]glutamate are employed as precursors. Both LA and DA also inhibit the uptake and increase the incorporation of ³²Pi into organophosphorus by etiolated barley leaves. These results indicate that LA and DA have more far-reaching effects upon plant metabolism than was previously believed.     

The initial metabolic conversion of levulinic acid in Cupriavidus necator

Levulinic acid or 4-ketovaleric acid is a potential renewable substrate for production of polyhydroxyalkanoates. In this work, the initial reactions of LA metabolism by Cupriavidus necator were examined in vitro. The organic acid was converted by membrane-bound crude enzymes obtained from the cells pre-grown on LA, while no LA activity was detected from cells pre-grown on acetic acid. Acetyl-CoA and propionyl-CoA were two major intermediates in the initial reactions of LA conversion. A mass balance on propionyl-CoA accounts for 84 mol% of LA added in vitro. It explains an interesting phenomenon that 3-hydroxbutyrate and 3-hydroxyvalerate are two major monomers of the biopolyester formed from LA, instead of 4-hydroxvalerate that has the similar chemical structure of LA as the precursor. A Monod model was used to describe the kinetics of LA utilization as a sole carbon source or a co-substrate of glucose and fructose. The μ_max and K_m of LA alone were 0.26 h⁻¹ and 0.01 g/L, respectively. The content and composition of PHA are also dependent on the culture conditions such as carbon to nitrogen ratio. The in vitro observation is supported by the high utilization rate of LA and the high molar percentage of 3HB and 3HV in the PHA derived from LA.     

Acknowledgment

The uptake and incorporation in vitro of various nucleic acid precursors by microfilariae, third-stage infective larvae, 10-day-old juveniles and adult worms of Brugia pahangi were investigated using scintillation counting and autoradiographic techniques. A significant uptake of uracil and of purines, including adenine, hypoxanthine, and guanine was demonstrated in this study. No evidence was obtained for the uptake and incorporation of thymine, cytosine, orotate, formate, folate or p-aminobenzoic acid by either micro- or macrofilariae of B. pahangi.     

Biosynthesis of riboflavine by a purine-requiring mutant strain of Escherichia coli

1. Riboflavine biosynthesis occurs in non-proliferating cultures of a purine-requiring strain of Escherichia coli (ATCC no. 13863). 2. No significant incorporation of radioactivity from [1-¹⁴C]glycine into either C-4a and C-9a of riboflavine or into nucleic acid purines is detected under the above conditions; appreciable incorporation of label into 5-aminoimidazole-4-carboxamide occurs. However, the label of [6-¹⁴C]guanine is incorporated significantly into C-4 of riboflavine and into nucleic acid adenine and guanine; the specific radioactivity of the riboflavine is approximately twice that of either adenine or guanine of nucleic acid. 3. These results show that a purine derivative is an obligatory intermediate in riboflavine biogenesis.     

A Role for Oxidized DNA Precursors in Huntington's Disease–Like Striatal Neurodegeneration

Several human neurodegenerative disorders are characterized by the accumulation of 8-oxo-7,8-dihydroguanine (8-oxodG) in the DNA of affected neurons. This can occur either through direct oxidation of DNA guanine or via incorporation of the oxidized nucleotide during replication. Hydrolases that degrade oxidized purine nucleoside triphosphates normally minimize this incorporation. hMTH1 is the major human hydrolase. It degrades both 8-oxodGTP and 8-oxoGTP to the corresponding monophosphates. To investigate whether the incorporation of oxidized nucleic acid precursors contributes to neurodegeneration, we constructed a transgenic mouse in which the human hMTH1 8-oxodGTPase is expressed. hMTH1 expression protected embryonic fibroblasts and mouse tissues against the effects of oxidants. Wild-type mice exposed to 3-nitropropionic acid develop neuropathological and behavioural symptoms that resemble those of Huntington''s disease. hMTH1 transgene expression conferred a dramatic protection against these Huntington''s disease–like symptoms, including weight loss, dystonia and gait abnormalities, striatal degeneration, and death. In a complementary approach, an in vitro genetic model for Huntington''s disease was also used. hMTH1 expression protected progenitor striatal cells containing an expanded CAG repeat of the huntingtin gene from toxicity associated with expression of the mutant huntingtin. The findings implicate oxidized nucleic acid precursors in the neuropathological features of Huntington''s disease and identify the utilization of oxidized nucleoside triphosphates by striatal cells as a significant contributor to the pathogenesis of this disorder.     

Entamoeba histolytica: Nucleic acid precursors affecting axenic growth

Treatment of Panmede and Trypticase, in water, with activated carbon and subsequently combining the filtered solution with glucose, cysteine, salts, serum and vitamins results in a medium depleted in the nucleic acid precursors required to sustain a high level of growth of axenic Entamoeba histolytica. Additions to the depleted medium which stimulated sustained growth, were the following, in the order of increasing efficacy: adenosine, adenine, AMP, AMP + GMP, AMP + GMP + UMP + CMP, or yeast ribonucleic acid. The last three additions restored growth to the level of cultures in TP-S-1 medium. No stimulation of sustained growth was found with GMP, IMP, UMP, or CMP, singly.     

Pyrimidine biosynthesis and its regulation in the estrogen-stimulated chick oviduct.

Studies on the incorporation of radio-labeled precursors into orotic acid and the pyrimidine nucleotides of RNA have established the occurrence of the orotate pathway for the de novo biosynthesis of pyrimidines in the chick oviduct. Measurements of the rate of incorporation of precursors into orotic acid in minces of oviduct revealed the activity of the orotate pathway to be accelerated in response to estrogen-stimulated nucleic acid synthesis and tissue growth. These data indicate that extrahepatic tissues of avian species meet their requirements for pyrimidine nucleotides through de novo synthesis rather than depend upon the liver or other exogenous sources for a supply of preformed pyrimidines. An examination of the influence of pyrimidine and purine nucleosides on the incorporation of radio-labeled precursors into orotic acid yielded evidence that pyrimidine biosynthesis in the chick is quite sensitive to inhibition by both purines and pyrimidines; the data indicate the reaction catalyzed by carbamoylphosphate synthetase to be the site of inhibition in both cases.     

Campylobacter jejuni Lipooligosaccharide Sialylation,Phosphorylation, and Amide/Ester Linkage Modifications Fine-tune Human Toll-like Receptor 4 Activation

Campylobacter jejuni is a leading cause of acute gastroenteritis. C. jejuni lipooligosaccharide (LOS) is a potent activator of Toll-like receptor (TLR) 4-mediated innate immunity. Structural variations of the LOS have been previously reported in the oligosaccharide (OS) moiety, the disaccharide lipid A (LA) backbone, and the phosphorylation of the LA. Here, we studied LOS structural variation between C. jejuni strains associated with different ecological sources and analyzed their ability to activate TLR4 function. MALDI-TOF MS was performed to characterize structural variation in both the OS and LA among 15 different C. jejuni isolates. Cytokine induction in THP-1 cells and primary monocytes was correlated with LOS structural variation in each strain. Additionally, structural variation was correlated with the source of each strain. OS sialylation, increasing abundance of LA d-glucosamine versus 2,3-diamino-2,3-dideoxy-d-glucose, and phosphorylation status all correlated with TLR4 activation as measured in THP-1 cells and monocytes. Importantly, LOS-induced inflammatory responses were similar to those elicited by live bacteria, highlighting the prominent contribution of the LOS component in driving host immunity. OS sialylation status but not LA structure showed significant association with strains clustering with livestock sources. Our study highlights how variations in three structural components of C. jejuni LOS alter TLR4 activation and consequent monocyte activation.     

Lipoic acid stimulates cAMP production via G protein-coupled receptor-dependent and -independent mechanisms

Lipoic acid (LA) is a naturally occurring fatty acid that exhibits anti-oxidant and anti-inflammatory properties and is being pursued as a therapeutic for many diseases including multiple sclerosis, diabetic polyneuropathy and Alzheimer's disease. We previously reported on the novel finding that racemic LA (50:50 mixture of R-LA and S-LA) stimulates cAMP production, activates prostanoid EP2 and EP4 receptors and adenylyl cyclases (AC), and suppresses activation and cytotoxicity in NK cells. In this study, we present evidence that furthers our understanding of the mechanisms of action of LA. Using various LA derivatives, such as dihydrolipoic acid (DHLA), S,S-dimethyl lipoic acid (DMLA) and lipoamide (LPM), we discovered that only LA is capable of stimulating cAMP production in NK cells. Furthermore, there is no difference in cAMP production after stimulation with either R-LA, S-LA or racemic LA. Competition and synergistic studies indicate that LA may also activate AC independent of the EP2 and EP4 receptors. Pretreatment of PBMCs with KH7 (a specific peptide inhibitor of soluble AC) and the calcium inhibitor (Bapta) prior to LA treatment resulted in reduced cAMP levels, suggesting that soluble AC and calcium signaling mediate LA stimulation of cAMP production. In addition, pharmacological inhibitor studies demonstrate that LA also activates other G protein-coupled receptors, including histamine and adenosine but not the β-adrenergic receptors. These novel findings provide information to better understand the mechanisms of action of LA, which can help facilitate the use of LA as a therapeutic for various diseases.