Euryhaline Chrysomonads: Nutrition and Toxigenesis in Prymnesium parvum, with Notes on Isochrysis galbana and Monochrysis lutheri

SYNOPSIS. The nutritional requirements of 3 isolates of Prymnesium parvum (2 Israeli, 1 Scottish) included vitamin B₁₂ and thiamine. For comparison, 2 other brackish chrysomonads were studied: Monochrysis lutheri isolated by Droop in Scotland and Isochrysis galbana purified by McLaughlin from a culture obtained from the Plymouth laboratory.
The isolates of Prymnesium parvum and Isochrysis galbana had a molecular B₁₂ specificity like Ochromonas malhamensis : no response to Factor B, pseudovitamin B₁₂, Factor A or Factor H. M. lutheri , in contrast, responded to pseudovitamin B₁₂, Factor H, and Factor A.
Thiamine was essential; 1.0 μg.% allowed full growth of P. parvum. The NaCl concentration for good growth was 0.3–5.0%; growth was possible from 6–12%. Dark growth was not achieved.
Ammonia, as suggested from its use in suppressing outbreaks of P. parvum , was sharply inhibitory, less so at high concentrations of NaCl or at acid pH.
Nitrate, ammonia, arginine, asparagine, methionine, histidine, alanine, glycine, serine, proline, leucine, isoleucine, tyrosine, aspartic and glutamic acids, acetylurea, and creatine served as nitrogen sources in both acid and alkaline media.
The phosphate requirement of P. parvum and M. lutheri and Isochrysis galbana was satisfied by inorganic phosphate, commercial glycerophosphate, yeast adenylic acid, cytidylic acid, monoethyl phosphate, and riboflavin monophosphate.
Laboratory cultures in defined media of the isolates of P. parvum were toxigenic to Lebistes and Gambusia. Culture fluids from alkaline media were more toxic than those from acid media, as previously noted in Israel.
Culture media suitable for production of large quantities of these organisms were developed.     

Growth inhibition of Candida albicans by folate pathway inhibitors. Their potential in the selection of auxotrophs

Growth studies were conducted on C. albicans in a glucose — salts — biotin (GSB) medium in the presence of folate inhibitors. Sulfanilamide inhibited growth which was restored by PABA or tetrahydrofolate (THF). Aminopterin inhibited growth to about the same level as did sulfanilamide, but this inhibition was not reversed with PABA nor THF, singly or in combination. Inhibition by combined sulfanilamide and aminopterin was synergistic, reducing growth by more than 90% for 48 h. The sulfanilamide component of the combined inhibition was reversed by PABA or THF to the level of that of aminopterin alone. Cytochrome synthesis was not affected by the inhibitors, but marked increases occurred in -ketoglutarate, malate, isocitrate, and pyruvate dehydrogenases, especially in the presence of both inhibitors. The pyrimidines in combination with sulfanilamide were as inhibitory as was the combination of aminopterin and sulfanilamide, but they had no effect when added alone or in combination with aminopterin. Unlike the pyrimidines, the purines stimulated about a 50% recovery from inhibition by either of the inhibitors. Growth inhibition by combined sulfanilamide and aminopterin was overcome by about 50% by the addition of the THF-mediated end-produits: deoxythymidylate, adenine, histidine and methionine. The use of GSB medium containing adenine, histidine, methionine and the folate inhibitors but without deoxythymidylate resulted in thymineless death of prototrophic cells providing a method for the selection of auxotrophic mutants.     

Formation of vitamins by pure cultures of tempe moulds and bacteria during the tempe solid substrate fermentation

The formation of water soluble vitamins (vitamin B₁₂, vitamin B₆, riboflavin, thiamine, nicotinic acid and nicotinamide) during the tempe solid substrate fermentation was investigated. The role of several strains of Rhizopus oligosporus, R. arrhizus , and R. stolonifer and the role of several bacteria in the vitamin formation process were checked. All fungal and bacterial strains were isolated from Indonesian tempe and soaking water samples. The Rhizopus strains formed riboflavin, nicotinic acid, nicotinamide and vitamin B₆. The final concentrations of these substances depended on the different strains involved and on the fermentation time. Isolates of R. oligosporus were generally the best vitamin formers. The moulds did not produce physiologically active vitamin B₁₂. The thiamine content decreased during fermentation. The addition of bacteria, which had been selected in a screening for vitamin B₁₂ production, resulted in an increase of physiologically active vitamin B₁₂. Citrobacter freundii and Klebsiella pneumoniae showed the best formation capabilities. Furthermore, the bacteria produced riboflavin and vitamin B₆ in addition to the moulds. The influence of Rhizopus on the vitamin B₁₂ formation of Cit. freundii was also investigated. The vitamin content of tempe that was fermented with the mould and the bacterium was three times as high as a control fermentation with Cit. freundii only.     

Regulation of nitrogen fixation by nitrite and glutamine in Derxia gummosa

Abstract Nitrogenase activity of cells of Derxia gummosa (30 h growth in cultures without combined nitrogen) was not inhibited on adding nitrate. However, on adding either azaserine or methionine sulfoximine (MSX) with nitrate to these cells, nitrogenase (C₂H₂ reduction) was inhibited because nitrite accumulated in the reaction mixtures. Nitrite inhibition of the in vivo C₂H₂ reduction had a K _i value of 16 μM. Both ammonia and glutamine inhibited N₂ fixation (C₂H₂ reduction) in intact cells and in those treated with toluene. This inhibition by ammonia was relieved by methionine sulfoximine but not by glutamine. Azaserine enhanced the inhibition of nitrogenase produced by either ammonia or glutamine, since these treatments resulted in an accumulation of glutamine.     

Functional impairment of rat Kupffer cells induced by aflatoxin B1 and its metabolites

Abstract Contamination of food with mycotoxins is a major health problem. Impairment of several immune functions has been repeatedly reported in animals fed with contaminated fodder. Since the liver is a major target of toxicity by aflatoxins, the effects of aflatoxins B1, and its hepatic metabolites Q₁ and M₁ on Kupffer cell function was investigated in vitro. Aflatoxin B₁ induced significant ( P < 0.05) inhibition of phagocytosis, intracellular killing of Candida albicans , and intrinsic anti-Herpes virus activity at concentrations as low as 0.01 pg ml⁻¹. Aflatoxin Q₁ and M₁ had similar effects on phagocytosis and microbicidal activity, but were two- to ten-fold less potent than aflatoxin B₁.     

Vitamin B12 Production by the Gastro-intestinal Microflora of Baboons Fed either a Vitamin B12 Deficient Diet or a Diet Supplemented with Vitamin B12

The vitamin B₁₂-producing capacity of micro-organisms isolated from baboon faeces and gastric contents was measured using Lactobacillus leichmannii . The animals were fed either a diet deficient in or supplemented with vitamin B₁₂ (controls). Samples of gastric and small intestinal contents, obtained at laparotomy from two young vitamin B₁₂-deprived baboons, contained varying quantities of vitamin B₁₂. Many of the organisms isolated from these aspirates produced vitamin B₁₂ in vitro . The highest levels of vitamin B₁₂ were produced by anaerobic organisms. Gastric juice samples from vitamin B₁₂ deprived and control baboons contained similar types of organisms with like vitamin B₁₂-producing capacity. The vitamin B₁₂ content, pH and total bacterial counts of gastric juice samples aspirated after a 6 h fast from vitamin B₁₂ deprived baboons were not significantly different from those of the control animals. The pH values of gastric juice samples aspirated 18 h after feeding, however, were significantly lower than those of 6 h fasting samples in both groups. The mean vitamin B₁₂ levels in the total volumes of gastric juice aspirated after each fasting period were similar. The possible involvement of the gastrointestinal flora in the vitamin B₁₂ status of the baboon is discussed.     

The influences of fungitoxicants on growth and aflatoxin production by Aspergillus flavus

The influence of six fungitoxicants on growth and aflatoxin production by Aspergillus flavus was tested in liquid SMKY medium at two concentrations, viz . 0.1 and 0.5%. Thiram completely inhibited the aflatoxin production at 0.5% concentration. Other fungitoxicants showing more than 60% inhibition were bavistin and daconil. Vitavax (0.1%) and agrosan GN (0.1 and 0.5%) stimulated the growth of fungus and aflatoxin elaboration after 7 d of incubation. Dithane M-45 moderately inhibited aflatoxin synthesis. Treatment with fungitoxicants also alters the ratio of B₁ and G₁.     

Copper and Iron as Determinant Factors of Antibiotic Production by Pseudomonas reptilivora

Production by Pseudomonas reptilivora of three antibiotics, named A, B₁ and B₂, took place in media containing either an inorganic or organic nitrogen source but only in the presence of available copper and iron. Higher yields were obtained in media containing inorganic nitrogen. Antibiotic synthesis was mediated by definite levels of copper and iron which determined the production of (a) the iron and copper-dependent activity A, (b) the two copper-dependent activities B₁ and B₂, or (c) a mixture of the three.     

Factors Mediating the Vitamin B12 Requirement of Euglena

SYNOPSIS. Deprived of vitamin B₁₂, Euglena gracilis strain Z ceases to divide which we believe to be a function of the light regime: division inhibition occurs more quickly in continuous light than in alternating (6L : 6D) light and not at all in total darkness. This phenomenon is dependent on the carbon source; cells grown in glutamate-malate medium do not divide regardless of the culture conditions while dl -lactate as carbon source permits growth in darkness in the absence of B₁₂. Conditions which lead to an increased O₂ or decreased CO₂ tension in the medium, such as agitation in darkness or incubation in red or white light, result in inhibition of division. This inhibition can be reversed by re-transferring the cells to still culture in the dark or, in the case of light-induced blockage, by the addition of DCMU.     

High Doses of Vitamin B6 in the Rat Are Associated with Inhibition of Hepatic Tryptophan Metabolism and Increased Uptake of Tryptophan into the Brain

Abstract: Acute administration of vitamin B₆ to rats (10 mg/kg body weight) led to reduced urinary excretion of N ¹-methyl nicotinamide and methyl pyridone carboxamide, indicating inhibition of the oxidative metabolism of tryptophan. There was a considerable reduction in the production of ¹⁴CO₂ from [ ring -2-¹⁴C]tryptophan, and a significant inhibition of hepatic tryptophan oxygenase when measured in liver homogenates, together with an increase in the concentration of tryptophan in plasma. There was an increase in both the concentration of tryptophan in the brain and the uptake into the brain of peripherally administered [³H]tryptophan, accompanied by a small increase in the rate of synthesis of 5-hydroxy-tryptamine in the brain. It is suggested that this increase in the uptake of tryptophan into the brain following a relatively large dose of vitamin B₆ may explain the beneficial action of the vitamin in some cases of depressive illness.     

The Role of Vitamin B12 Binding In the Uptake of the Vitamin By Euglena Gracilis

The uptake of [⁵⁷Co]B₁₃ (cyanocobalamin) by Euglena gracilis strain Z (ATCC 12716) occurred in 2 distinct phases-an initial rapid phase followed by a slower secondary phase. This secondary phase appeared after the saturation of the binding sites involved in the initial rapid phase and was energy-dependent and completely inhibited by 2,4-dinitrophenot, KCN and sodium azide. the subcellular localization of labeled cyanocobalamin taken up by the cell was mostly contained in the chloroplast fraction. the time course and the saturation kinetics of B₁₂ uptake by purified chloroplast fraction indicated that this fraction and the intact cell had a similar affinity for the vitamin B₁₂. This suggested that the chloroplasts contained the binding sites for vitamin ₁₂ and might regulate the uptake process in the intact cell. the kinetic properties of the overall ₁₂ uptake mechanism suggested that the initial phase represent the binding of vitamin ₁₂ to the available sites on the chloroplast. the secondary phase may represent the de novo synthesis of new binding sites.     

Lipid Composition of the Brain in the Vitamin B12-Deficient Fruit Bat (Rousettus aegyptiacus) with Neurological Impairment

Abstract: The Egyptian fruit bat Rousettus aegyptiacus develops severe vitamin B₁₂ deficiency when fed a diet of fresh peeled fruit and water. In a group of bats fed this diet, B₁₂ concentrations in the serum and brain were low, and neurological impairment, evidenced by deficient or absent hindlimb groping or grasping ability and climbing difficulties, was manifest. Control bats fed the identical diet supplemented with B₁₂ showed no such changes. Fatty acid analysis of whole brain homogenates showed a higher level of 20:4 in the deficient group. Phosphatidylcholine showed a marginally higher percentage of 18:3. The total percentage of branched chain fatty acids of phos-phatidylethanolamine was four times higher in deficient brains, comprising 2% of the total. Sphingomyelin showed a slightly higher percentage of 15:0, and a significantly lower percentage of long chain fatty acids C-24 and longer ( p < 0.01). The compositions of nonhydroxy fatty acids in cerebroside were unchanged. Examination of phospholipids showed that 8.9 ± 0.4% of total phosphorus was present as sphingomyelin in deficient bats, compared with 11.9 ± 1.2% in control animals (p < 0.05). There were no statistically significant differences in the concentrations of total brain lipid, protein, phospholipid, glycosphingolipid, cholesterol and plasmalogen between B₁₂-deficient and control bats.     

Synchronization of Division in Vitamin B12-Starved Euglena gracilis

SYNOPSIS Vitamin B₁₂ deficiency arrests cell division in Euglena gracilis. B₁₂ starvation for short periods made it possible to induce synchronous growth by addition of the vitamin. Culture conditions were established to optimize replenishment synchrony. The DNA content of E. gracilis in steady state culture and vitamin B₁₂ deficiency culture was measured by flow cytofluorometry and was consistent with colorimetric determinations. The cell volume and DNA distributions of E. gracilis in synchronous culture were analyzed and the sequential changes during the division cycle were computed. Synchronous culture permits more definitive studies of shifts in cell volume and DNA distributions, in which the biochemical events required for cell division are presumably synchronized.     

VITAMIN B6 TRANSPORT IN THE CENTRAL NERVOUS SYSTEM: IN VIVO STUDIES

Abstract— The total concentrations of vitamin B₆ (B₆) in plasma, choroid plexus, CSF and brain of adult New Zealand white rabbits, measured fluorometrically, were 0.30, 15.10, 0.39 and 8.90 μ mol/l or kg respectively. The mechanisms by which B₆ enters and leaves brain, choroid plexus and CSF were investigated by injecting [³H]pyridoxine (PIN) intravenously, intraventricularly and intraarterially. [³H]PIN, with or without unlabelled PIN, was infused intravenously at a constant rate into conscious rabbits. At 150 min, [³H]B₆ readily entered CSF, choroid plexus and brain. The addition of 0.5 mmol/kg carrier PIN to the infusion solution depressed the relative entry of [³H]B₆ into CSF, choroid plexus and brain by about 80%. After intraventricular injection, [³H]PIN readily entered brain from CSF. The intraventricular injection of carrier PIN with [³H]PIN decreased the amount of [³H]B₆ in brain and also decreased the percentage of [³H]B₆ in CSF and brain that was phosphorylated. During one pass through the cerebral circulation, [³H]PIN (1 μ m ) was cleared from the circulation no more rapidly than mannitol. These results were interpreted as showing that the entry of B₆ from blood into CSF and presumably the extracellular space of brain and thence into brain cells involves one or more saturable transport and/or metabolic steps.     

Lactobacillus reuteri CRL 1098 prevents side effects produced by a nutritional vitamin B12 deficiency

Aims:  To evaluate the efficiency of the vitamin B_12-producing Lactobacillus reuteri CRL1098 strain in preventing the symptoms caused by a nutritional cobalamin-deficient diet in pregnant female mice and their weaned offspring.
Methods and Results:  Pregnant female mice were divided into three groups: animals fed with a B₁₂-deficient diet (DD), animals fed with DD plus L. reuteri CRL1098 and animals fed with a B₁₂-sufficient diet. The animals received the different feedings from the end of gestation up to weaning. At the end of the trials, they and their corresponding offspring were bled to determine haematological, immunological and histological parameters. The administration of the pseudovitamin B₁₂-producing strain prevented the symptoms observed in female and weaned young animals fed with a nutritional B₁₂-deficient diet.
Conclusions:  Our data suggest that the pseudovitamin B₁₂ produced by L. reuteri CRL1098 is biologically active and effective in preventing the pathologies caused by the nutritional deficiency of B₁₂ both in pregnant mice and their offspring.
Significance and Impact of the Study:  The ability of L. reuteri CRL1098 to prevent a nutritional vitamin deficiency was demonstrated for the first time. The addition of a GRAS micro-organism to complement the B₁₂ content in deficient foods is an interesting biotechnological alternative.     

Influence of microbial co-inhabitants on aflatoxin synthesis of Aspergillus flavus on maize kernels

The co-inhabiting mycoflora with Aspergillus flavus observed on individual maize kernels was evaluated for its influence on aflatoxin synthesis. All 13 types of associations of different fungal species inhibited aflatoxin B₁ and G₁ production at different levels (34·3–100%). Inhibition of radial growth of A. flavus by Fusarium moniliforme (59·8%), Trichoderma viride (72·5%) and Rhizopus nigricans (42%) could be directly correlated to the per cent inhibition of aflatoxin production. High levels of inhibition of aflatoxin elaboration were noted in competition of A. flavus with other toxigenic moulds.     

B1 and B2 Bradykinin Receptors Encoded by Distinct mRNAs

Abstract: Bradykinin receptors have been subdivided into at least two major pharmacological subtypes, B₁ and B₂. The cDNAs encoding functional B₂ receptors have recently been cloned, but no molecular information exists at present on the B₁ receptor. In this article, we describe experiments examining the possible relationship between the mRNAs encoding the B₁ and B₂ types of receptor. We showed previously that the Human fibroblast cell line W138 expresses both B₁ and B₂ receptors. In this report, we describe oocyte expression experiments showing that the B₁ receptor in W138 human fibroblast cells is encoded by a distinct mRNA ∼2 kb shorter than that encoding the B₂ receptor. We have used an antisense approach in conjunction with the oocyte expression system to demonstrate that the two messages differ in sequence at several locations throughout the length of the B₂ sequence. Taken together with the mixed pharmacology exhibited in some expression systems by the cloned mouse receptor, the data indicate that B₁-type pharmacology may arise from two independent molecular mechanisms.     

SOME EFFECTS OF DIETARY VITAMIN B6 DEFICIENCY AND 4-DEOXYPYRIDOXINE ON γ-AMINOBUTYRIC ACID METABOLISM IN RAT BRAIN

Abstract— Investigations of the respective effects of dietary vitamin B₆ deficiency and 4-deoxypyridoxine (a vitamin B₆ antagonist) on GABA metabolism in rat brain have been carried out. No convulsions were observed in rats subjected to either treatment. GABA levels were lowered by both treatments, the greatest diminutions being found with the dietary deficiency. Glutamic acid decarboxylase activity was reduced under both conditions, but the loss of activity in the B₆ deficiency experiments could be attributed to cofactor depletion, whereas in the deoxypyridoxine experiments the loss of activity appears to be due to lower levels of available apoenzyme. The activity of GABA-transaminase was not affected by deoxypyridoxine treatment and only moderately reduced in the B.5 deficient animals.     

Enhancement of vitamin B6 levels in seeds through metabolic engineering

As a versatile cofactor for many enzymes catalyzing important biochemical reactions, vitamin B₆ is required for all cellular organisms. In contrast to bacteria, fungi and plants, which have the ability to synthesize vitamin B₆ de novo , animals have to take up the vitamin from their diet. Plants are the major source of vitamin B₆ for animals. The recent identification of vitamin B₆ biosynthetic enzymes PDX1 and PDX2 in plants makes it possible to regulate the biosynthesis of this important vitamin. In this study, we generated Arabidopsis plants overexpressing the PDX1 and/or PDX2 gene and used a liquid chromatography/mass spectrometry/mass spectrometry method to determine the levels of different forms of vitamin B₆ in these transgenic plants. It was found that expression of the PDX genes under control of the CaMV 35S promoter caused only a limited increase in pyridoxine contents in dry seeds but not in shoots or roots. When using the Arabidopsis seed-specific 12S promoter to drive the expression of the PDX genes, the levels of vitamin B₆ increased more than twofold in transgenic plants. Our work demonstrates that it is feasible to enhance vitamin B₆ content in seeds by metabolic engineering.