Cryopreservation of economically valuable marine micro-algae in the classes Bacillariophyceae, Chlorophyceae, Cyanophyceae, Dinophyceae, Haptophyceae, Prasinophyceae, and Rhodophyceae

The ability to routinely cryopreserve micro-algal species reduces costs associated with maintaining large culture collections and reduces the risks of losing particular strains or species through contamination and genetic drift. Cryopreservation is also a useful adjunct in aquaculture hatcheries for strains of micro-algae where the nutritional status may change as a result of continuous sub-culture. In this study, cryopreservation of isolates from seven micro-algal classes was investigated. Successful candidates included the marine dinoflagellates Amphidinium carterae, Amphidinium trulla, and Gymnodinium simplex, and the haptophytes Chrysochromulina simplex, Prymnesium parvum, Prymnesium parvum f. patelliferum, Isochrysis galbana, and Pavlova lutheri. Also successfully cryopreserved were the planktonic diatoms Chaetoceros calcitrans, Chaetoceros muelleri, Chaetoceros sp., and the benthic Nitzschia ovalis, the chlorophyte Chlamydomonas coccoides, the rhodophyte Porphyridium purpureum, the prasinophytes Tetraselmis chuii, and Tetraselmis suecica, and the cyanophytes Raphidiopsis sp., and Aphanizomenon flos-aquae. All species were successfully cryopreserved using 15% Me2SO.     

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.     

Specificity of Glycerol for Dark Growth of Prymnesium parvum

SYNOPSIS. Out of 64 nutrients tested, none replaced glycerol for growth in the dark of Prymnesium parvum. Some metabolites enahanced growth in the light, and in the presence of glycerol also in the clark. Good growth with glycerol could be obtained in the absence of CO₂. Survival of cultures in the dark in media without glycerol was prolonged by various nutrients. Thioglycerol in glycerol-containing media inhibited growth in light and darkness. Apparently Prymnesium parvum has a specific glycerol requirement for dark growth.     

Fumonisin B1 production by Fusarium proliferatum strains isolated from Allium fistulosum plants and seeds in Japan

Aims:  To test the fumonisin B₁ - producing ability of Fusarium proliferatum strains isolated from Welsh onion ( Allium fistulosum ) plants and seeds of commercial cultivars in Japan and to examine the applicability of PCR-based assays to discriminate between fumonisin B₁-producing and nonproducing isolates.
Methods and Results:  Fumonisin B₁ levels in 20 Fusarium isolates obtained from Welsh onion plants and seeds of seven commercial cultivars were determined by HPLC. Thirteen of the 20 isolates produced fumonisin B₁. PCR assay with FUM1 gene-specific primers amplified a DNA fragment (700 bp) only from fumonisin-producing isolates.
Conclusions:  Fusarium proliferatum isolates that can produce fumonisin B₁ were often associated with wilted Welsh onion plants and seeds of some commercial cultivars. The PCR assay with FUM1 gene-specific primers has the potential to discriminate between fumonisin B₁-producing and nonproducing isolates.
Significance and Impact of the Study:  This study revealed that F. proliferatum producing fumonisin B₁ is associated with Welsh onion plants and that commercial cultivar seeds may be contaminated with the fungus. PCR amplification of FUM1 gene can be a useful tool for the rapid identification of fumonisin B₁-producing F. proliferatum isolates.     

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.     

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.     

COMPARATIVE STUDIES ON ALDOLASE ACTIVITY IN MARINE PLANKTONIC ALGAE,AND THEIR EVOLUTIONARY SIGNIFICANCE1

Fructose diphosphate aldolase activity was examined in acetone powders and cell-free extracts of 15 photoautotrophically grown marine planktonic species belonging to 6 algal divisions as follows: Chlorophyta: Tetraselmis maculata, Dunaliella tertiolecta; Chrysophyta: Monochrysis lutheri, Isochrysis galbana, Prymnesium parvum, Coccolithus huxleyi; Bacillariophyta: Phaeodactylum tricornutum, Skeletonema costatum, Cyclotella nana; Cryptophyta: Cryptomonas sp., Rhodomonas lens, Hemiselmis virescens; Pyrrophyta: Amphidinium carteri; Cyanophyta: Anacystis marina, Agmenellum quadruplicatum. Indications of the types of aldolase (Rutter's classes) present in each alga were obtained from comparative studies of the effects of pH and of the following reagents on the activity: ethylenediamine tetraacetate, dithiothreitol, p-chloromercuriphenyl sulfonate. Type I (higher plant-animal type) aldolase only was indicated in the 2 chlorophytes, in I chrys-ophyte (M. lutheri), and in 1 bacillariophyte (P. tricornutum), while the remaining algae appeared to contain either exclusively or principally Type II (bacterial-fungal type) aldolase. The evolutionary implications of these findings are discussed.     

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.     

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.     

Action of Sulfanilamide on Ochromonas malhamensis

SYNOPSIS. Sulfanilamide inhibited the growth of O. malhamensis. Sulfanilamide growth inhibition was reversed competitively by PABA and by very high concentrations of folic acid. Folic acid at low concentrations, however, accentuated sulfa inhibition of growth. Vitamin B₁₂, methionine, p -aminobenzoylglutamic acid and pteroic add were effective to some extent as antagonists of sulfa. A marked reduction in the folate synthesis was accompanied by sulfa growth inhibition. This was restored on growth restoration by PABA, folic acid, vitamin B₁₂ and methionine. The reduction in folate synthesis held for all the folate fractions except one derivative—a formyl poly-glutamate. Sulfanilamide-inhibited cells had a considerable activity for in vitro synthesis of folate activity from precursors. ∼75% activity being retained at the 90% growth inhibition level. There was no change in chlorophyll, RNA and DNA contents as a result of sulfa growth inhibition.     

Effect of water activity and temperature on growth and fumonisin B1 and B2 production by Fusarium proliferatum and F. moniliforme on maize grain

S. MARIN, V. SANCHIS, I. VINAS, R. CANELA AND N. MAGAN. 1995. The effect of different water activities ( a _w, 0.968, 0.956, 0.944, 0.925) and temperature (25°C and 30°C) on colonization and production of fumonisin B₁ (FB₁) and B₂ (FB₂) on sterile layers of maize by Fusarium proliferatum and F. moniliforme isolates was determined over periods of 6 weeks. Generally, both F. moniliforme and F. proliferatum grew faster with increasing a _w and best at 30°C. All three isolates produced more FB 1 than FB₂ regardless of a _w or temperature. Very little FB₁ and FB₂ were produced at 0.925 a _w, with maximum produced at 0.956 and 0.968 a _w at both temperatures tested. Most FB₁ and FB₂ were produced by F. moniliforme (25N), followed by F. proliferatum isolates (73N and 131N). At all a _w levels and both temperatures there was an increase in FB₁ and FB₂ concentration with time. Statistical analyses of a _w, temperature, time, two- and three-way interactions showed some significant differences between isolates and FB₁ and FB₂ production.     

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.     

Utilization of dissolved inorganic carbon (DIC) and the response of the marine flagellate Isochrysis galbana to carbon or nitrogen stress

The growth of the marine flagellate Isochrysis galbana was followed in batch cultures at four concentrations of dissolved inorganic carbon (DIC), from C- and N-replete lag phase into C- and/or N-deplete stationary phase. Organic buffers were omitted from the growth medium, and culture pH was maintained at 8.30±0.05 by the addition of acid or alkali. The responses of the flagellate to N stress included an increase in the C:N ratio, and decreases in the ratios of glutamine (Gln):glutamate (Glu) and Chl a :C, and the cell Chl a quota. Conversely, the responses to C stress included a decrease in the C:N ratio, and increases in the ratios of Gln:Glu and Chl a :C, and the cell Chl a quota. The relationship between carbon-specific growth rate (C-μ), and the concentration of extracellular DIC, [DIC]_ext, exhibited Michaelis–Menten type kinetics with a half saturation constant, K _G(DIC), of 81 μM. Comparative studies of the diatom Phaeodactylum tricornutum showed similar results, although the value of K _G(DIC) was lower at 30 μM.     

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.     

Rescue by vitamin B12 of Escherichia coli cells treated with colicins A and E allows measurement of the kinetics of colicin binding on BtuB

Abstract Sensitivity of Escherichia coli bacteria to colicins A and E1 was significantly increased by overproduction of the BtuB receptor protein. The amount of vitamin B₁₂ needed before colicins A and E1 treatment to protect cells against killing was found to be a function of the number of BtuB molecules present at the cell surface. Cells treated by colicins A and E were rescued from killing by addition of vitamin B₁₂ shortly after colicin treatment. The rate of reversal by vitamin B₁₂ may correspond to the kinetics of irreversible binding to BtuB of the various colicins.     

SUBCELLULAR DISTRIBUTION OF B6 VITAMERS IN CEREBRAL CORTEX

Abstract— The distribution of B₆ vitamers in subcellular fractions of cerebral cortex was examined. No pyridoxine and only traces of pyridoxamine could be detected in cerebral cortex. Significant quantities of pyridoxal were found in the cytoplasmic fraction. No vitamin B₆ was detected in the subcellular fractions carrying microsomes, myelin, vesicles, and small membranes settling above the 1-0 M-sucrose gradient. Pyridoxamine phosphate was the predominant form of vitamin B₆ in cerebral cortex of mature rats and guinea pigs, being present in almost twice the concentration of pyridoxal phosphate. More of the latter compound was present in the cytoplasm than in the mitochondria. In contrast, pyridoxamine phosphate was compartmentalized in extraterminal and intraterminal mitochondria. Of especial interest was the finding that there was a significant amount of pyridoxamine phosphate attached to the presynaptic membrane and a small but detectable amount in the fraction containing postsynaptic membranes.     

癸二烯醛对3种常见浮游植物生长和光合作用的影响

多不饱和醛是硅藻细胞损伤后分泌的有机毒素, 能够抑制桡足类的生殖和幼体发育, 继而降低浮游动物对硅藻的摄食压力。但是有关多不饱和醛对浮游植物的毒性效应研究较少。选取东海原甲藻(Prorocentrum donghaiense)、双突角毛藻(Chaetoceros didymus)和小普林藻(Prymnesium parvum)进行毒性实验, 将处于指数生长期的藻细胞置于不同浓度的癸二烯醛中, 观察浮游植物在细胞生长及光合作用方面对癸二烯醛的响应, 从而评估多不饱和醛对藻类的影响。结果表明, 浓度为1 mg·L^-1的癸二烯醛即可对东海原甲藻产生显著的抑制作用; 当癸二烯醛浓度达到5 mg·L^-1时, 东海原甲藻和双突角毛藻细胞的生长以及光合作用均受到极显著抑制; 当癸二烯醛浓度达到10 mg·L^-1时, 3种浮游植物的生长完全受到抑制, 细胞密度、F_v/F_m、Yield值以及rETR均在24小时内降至0。研究结果表明, 癸二烯醛显著抑制了浮游植物的生长及光合作用, 但是不同藻类对癸二烯醛的毒性响应存在种间差异。多不饱和脂肪醛的产生有利于硅藻在营养盐缺乏时抑制其它浮游植物的生长, 从而保证自身的生物量积累。     

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.     

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.