Dehydroascorbic acid uptake and intracellular ascorbic acid accumulation in cultured Müller glial cells (TR-MUL)

Vitamin C is mainly transported across the inner blood–retinal barrier (inner BRB) as dehydroascorbic acid (DHA) via a facilitative glucose transporter (GLUT) 1, and accumulates as ascorbic acid (AA) in the retina. Müller cells, huge glial cells, exhibit passive structural and metabolic functions for retinal neurons and the inner BRB. We characterized DHA transport and its corresponding transporter in a rat Müller cell line (TR-MUL5 cells). [¹⁴C]DHA uptake by TR-MUL5 cells took place in a time-dependent and Na⁺-independent manner. [¹⁴C]DHA uptake was inhibited by substrates and inhibitors of GLUTs, suggesting that Müller cells take up DHA via GLUTs. HPLC analysis revealed that most of the DHA taken up by TR-MUL5 cells was converted to AA and accumulated as AA in TR-MUL5 cells. [¹⁴C]DHA uptake by TR-MUL5 cells took place in a concentration-dependent manner with a Michaelis–Menten constant of 198 μM and was inhibited by cytochalasin B in a concentration-dependent manner with a 50% inhibition concentration of 0.283 μM. Although GLUT1, 3, and 4 mRNA are expressed in TR-MUL5 cells, quantitative real-time PCR revealed that GLUT1 mRNA expression was 5.85- and 116-fold greater than that of GLUT3 and 4, respectively. Western blot analysis supports the expression of GLUT1 protein with 45 kDa in TR-MUL5 cells. In conclusion, DHA is taken up by facilitative glucose transporters, most likely GLUT1, and converted to AA in TR-MUL5 cells.     

Colchicine, nocodazole and trifluralin: different effects of microtubule polymerization inhibitors on the uptake and migration of endosymbiotic algae in Hydra viridis

Hydra viridis (= Chlorohydra viridissima) the freshwater coelenterate, is symbiotic. Each individual animal harbours Chlorella sp. in its endodermal cells. The symbiosis may be disestablished, the partners grown independently, and then re-established experimentally. The most effective method to produce alga-free hydra was developed by Pardy (1976). In this study algae from homogenized H. viridis, English (= European) strain, were either used directly or first grown in pure culture and then injected to re-establish the symbiosis. The uptake of algae grown in culture was compared with that of algae released directly from fresh hydra. Fewer cultured algae were taken up. Four strains were tested: English, Carolina, Frome, and Jubilee. Each takes up a characteristic number of Chlorella when injected in excess. Colchicine, nocodazole and trifluralin, were tested for their effects on either uptake or migration. Colchicine had no effect. Nocodazole and trifluralin reduced both the number of algae taken up and the rate at which they were transported to the distal region of the hydra digestive cell. The effects of these drugs tended to be proportional to the concentration between 10(-8) and 10(-5) M.     

Mechanism of proline uptake by Chlorella vulgaris

An amino acid uptake system specific for glycine, alanine, serine and proline was induced by glucose in Chlorella vulgaris. The uptake system translocated the zwitterionic form of the amino acid. There was more than 100-fold accumulation which indicated a coupling to metabolic energy. The depolarization of the membrane potential during proline uptake and the sensitivity of its uptake rate to the membrane potential point to coupling with an ion flow. Inhibitors of plasmalemma-bound H⁺-ATPase inhibit proline uptake. These data are interpreted to mean that proline is taken up as a proton symport. In some Chlorella strains the proline-coupled H⁺ uptake could be measured with electrodes, but not in Chlorella vulgaris. There is evidence that the transport of amino acids rapidly stimulates the proton-translocating ATPase of Chlorella vulgaris, so that the proline-coupled proton uptake is immediately neutralized.     

Fish oil fatty acids impair VLDL assembly and/or secretion by cultured rat hepatocytes

We determined the effect of the two major fish oil fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on VLDL assembly and secretion by cultured rat hepatocytes. The incorporation of [3H]glycerol into total triglyceride (cell plus media) was stimulated eight-fold when hepatocytes were incubated for 2 h with 1 mM EPA, DHA, or oleic acid (OA), suggesting that fish oil fatty acids stimulate hepatic triglyceride synthesis to an extent similar to OA. In contrast, mass quantitation of secreted triglyceride showed impaired triglyceride secretion with EPA and DHA compared to OA. During a 42-h time course, cells stimulated with EPA and DHA progressively accumulated triglyceride compared to cells stimulated with OA. To determine whether fish oil fatty acids impair very low density lipoprotein (VLDL) secretion, cells were labeled with [35S]methionine and the secretion of de novo synthesized apoB was measured. Compared to OA, EPA and DHA significantly impaired the secretion of both molecular weight forms of apoB. The cellular content of apoB was not altered by any of the fatty acids. The concordant decrease in the secretion of both triglyceride and apoB suggests that fish oil fatty acids impair VLDL assembly and/or secretion.     

Ascorbate transport from the apoplast to the symplast in intact leaves

Infiltration of reduced ascorbate (ASC) into the leaves of Betula pendula Roth and subsequent measurement of its loss therein after incubation allowed us to follow ascorbate transport from apoplast to symplast in intact leaves. All of the ascorbate extracted from the native apoplast was in fully oxidized form, dehydroascorbate (DHA). When 5 m M of ASC was infiltrated into the leaves, its intense decay occurred, but only 55% of ASC lost was recovered in apoplast as DHA. When ASC was added to the freshly extracted intercellular washing fluid (IWF), ASC oxidation occurred as well. However, all oxidized ASC was recovered as DHA, indicating that further decomposition of DHA did not occur. Similarly, all of the ASC infiltrated into the leaves was found therein either as ASC or DHA after incubation of leaves for up to 60 min. On this base the ascorbate infiltrated into the leaves and not recovered in the IWF was interpreted as ascorbate taken up into the symplast. The calculated uptake rates of ascorbate at different ASC concentrations followed saturation kinetics with the maximum uptake rate of 300 nmol m⁻² plasma membrane (PM) area min⁻¹ and Michaelis constant of 12.8 m M . The uptake of ascorbate was significantly inhibited by the addition of dithiothreitol or by PM H⁺ ATPase inhibitor erythrosin B. Thus, our results support the previous observations that DHA is preferably transported from the apoplastic to the cytoplasmic side of the membrane and show that this process is dependent upon PM proton gradient.     

Genotype versus phenotype variability in Chlorella and Micractinium (Chlorophyta, Trebouxiophyceae)

The most recent revision of the genus Chlorella, based on biochemical and SSU rDNA analyses, suggested a reduction to a set of four "true" spherical Chlorella species, while a growing number of morphologically different species such as Micractinium (formerly Micractiniaceae) were found to cluster within the clade of "true"Chlorella. In this study, the generic concept in Chlorellaceae to Chlorella and Micractinium was evaluated by means of combined SSU and ITS-2 rDNA sequence analyses and biotests to induce development of bristles on the cell wall. Molecular phylogenetic analyses of Chlorella and Micractinium strains confirmed their separation into two different genera. In addition, non-homoplasious synapomorphies (NHS) and compensatory base changes (CBC) in the secondary structures of SSU and ITS-2 rDNA sequences were found for both genera using this approach. The Micractinium clade can be differentiated into three different genotypes. Using culture medium of the rotifer Brachionus calyciflorus, phenotypic plasticity in Chlorella and Micractinium was studied. Non-bristled Micractinium cells developed bristles during incubation with Brachionus culture medium, whereas Chlorella did not produce bristles. Grazing experiments with Brachionus showed the rotifer preferred to feed on non-bristled cells. The dominance of colonies versus solitary cells in the Micractinium culture was not correlated with the "Brachionus factor". These results suggest that morphological characteristics like formation of bristles represent phenotypic adaptations to the conditions in the ecosystem.     

Enhanced incorporation of docosahexaenoic acid in serum, heart, and brain of rats given microemulsions of fish oil

Long-chain n-3 fatty acids are essential for the development of cognitive functions and reducing the risk factor for cardiovascular diseases. The present study was undertaken to prepare fish oil (FO) microemulsion and explore the possibility of enhancing the enrichment of long-chain n-3 PUFA in the heart and brain lipids. The bioavailability of encapsulated FO was compared with that of native oil in rats by utilizing the intestinal sac method and by an in vivo study giving microemulsions of FO through intubation for a period of 30 days. Microemulsions were prepared using chitosan, gum acacia, whey protein, and lipoid. The bioavailability of eicosapentaenoic acid and docosahexaenoic acid (DHA) from FO encapsulated in chitosan, gum acacia, whey protein, and lipoid was increased by 7, 9, 23, and 68 %, respectively, as compared to oil given without encapsulation in the everted intestinal sacs model. The DHA levels in serum lipids when FO was given as lipoid emulsion to rats were found to be 56 μg/ml, while rats given FO without encapsulation had a DHA level of 22 μg/ml. In the heart and brain lipids, the DHA levels were increased by 77 and 41 %, respectively, in rats given FO encapsulated with lipoid compared to those given native oil. These studies indicated that DHA from FO was taken up in a more efficient manner when given in an encapsulated form with lipoid. Thus, phospholipid-based binding materials such as Lipoid provide a good delivery system for FO and significantly enhance DHA levels in the serum, liver, heart, and brain tissues.     

Metabolism of Urea by Chlorella vulgaris

Urea metabolism was studied with nitrogen-starved cells of Chlorella vulgaris Beijerinck var. viridis (Chodat), a green alga which apparently lacks urease. Incorporation of radioactivity from urea-(14)C into the alcohol-soluble fraction was virtually eliminated in cell suspensions flushed with 10% CO(2) in air. This same result was obtained when expected acceptors of urea carbon were replenished by adding ornithine and glucose with the urea. Several carbamyl compounds, which might be early products of urea metabolism and a source of the (14)CO(2), were not appreciably labeled. If cells were treated with cyanide at a concentration which inhibited ammonia uptake completely and urea uptake only slightly, more than half of the urea nitrogen taken up was found in the medium as ammonia. Cells under nitrogen gas in the dark were unable to take up urea or ammonia, but the normal rate of uptake was resumed in light. Since 3-(3,4-dichlorophenyl)-1,1-dimethylurea did not selectively inhibit this uptake, an active respiration supported by light-dependent oxygen evolution in these cells was ruled out. A tentative scheme for urea metabolism is proposed to consist of an initial energy-dependent splitting of urea into carbon dioxide and ammonia. This reaction in Chlorella is thought to differ from a typical urease-catalyzed reaction by the apparent requirement of a high energy compound, possibly adenosine triphosphate.     

Docosahexaenoic acid metabolism and effect on prostacyclin production in endothelial cells

Bovine aortic endothelial cultures readily take up docosahexaenoic acid (DHA). Most of the DHA was incorporated into phospholipids, primarily in ethanolamine and choline phosphoglycerides, and plasmalogens accounted for 34% of the DHA contained in the ethanolamine fraction after a 24-h incubation. The retention of DHA in endothelial phospholipids was not greater than other polyunsaturated fatty acids and unlike arachidonic and eicosapentaenoic acids, DHA did not continue to accumulate in the ethanolamine phosphoglycerides after the initial incorporation. About 15% of the [14C(U)]DHA uptake was retroconverted to docosapentaenoic and eicosapentaenoic acids in 24 h. Some of the newly incorporated [14C(U)]DHA was released when the cells were incubated subsequently in a medium containing serum and albumin. The released radioactivity was in the form of free fatty acid and phospholipids and after 24 h, 11% was retroconverted to docosapentaenoic and eicosapentaenoic acids. Total DHA uptake was decreased only 10% by the presence of a 100 microM mixture of physiologic fatty acids, but as little as 10 microM docosatetraenoic acid reduced DHA incorporation into phospholipids by 25%. DHA was not converted to prostaglandins or lipoxygenase products by the endothelial cultures. When DHA was available, however, less arachidonic acid was incorporated into endothelial phospholipids, and less was converted to prostacyclin (PGI2). Enrichment of the endothelial cells with DHA also reduced their capacity to subsequently produce PGI2. These findings indicate that endothelial cells can play a role in DHA metabolism and like eicosapentaenoic acid, DHA can inhibit endothelial PGI2 production when it is available in elevated amounts.     

The determination of the membrane ptoential of Chlorella vulgaris. Evidence for electrogenic sugar transport.

From data on the accumulation of tetraphenylphosphonium within Chlorella vulgaris cells, it can be estimated that these cells possess a membrane potential of --120 to --150 mV (inside negative). Under anaerobic conditions as well as in the presence of uncoupling agents the membrane potential drops to about -60 to -80 mV. Nystatin (50 mug/ml) abolishes it almost completely. Since it took more than 1 h before the tetraphenylphosphonium equilibrium was reached, this method could not be used to measure relatively fast transient changes in membrane potential. However, the rate of influx of tetraphenylphosphonium is also directly dependent on membrane potential and can be followed within minutes. Using this phenomenon as an indicator for membrane potential a brief transient depolarisation was detected after the addition of sugars taken up by Chlorella via the proton cotransport system. The depolarisation was absent from cells not induced for sugar uptake and induced cells did not show it with substances not transported, like mannitol. The maximal depolarisation observed amounted to about 70 mV; after 1 min, however, the membrane potential returned to a value about 25 mV less negative than the one before sugars was added. The results demonstrate that sugar uptake in Chlorella is electrogenic. The delta pH plus membrane potential measured for Chlorella completely cover the energy required to explain the 1600-fold accumulation of 6-deoxyglucose experimentally observed.     

Plasma membrane gamma-glutamyltransferase activity facilitates the uptake of vitamin C in melanoma cells

Adequate cellular transport of ascorbic acid (AA) and its oxidation product dehydroascorbate (DHA) is assured through specific carriers. It was shown that vitamin C is taken up as DHA by most cell types, including cancer cells, via the facilitative GLUT transporters. Thus, AA oxidation to DHA can be considered a mechanism favoring vitamin C uptake and intracellular accumulation. We have investigated whether such an AA-oxidizing action might be provided by plasma membrane gamma-glutamyltransferase (GGT), previously shown to function as an autocrine source of prooxidants. The process was studied using two distinct human metastatic melanoma clones. It was observed that the Me665/2/60 clone, expressing high levels of membrane GGT activity, was capable of effecting the oxidation of extracellular AA, accompanied by a marked increase of intracellular AA levels. The phenomenon was not observed with Me665/2/21 cells, possessing only traces of membrane GGT. On the other hand, AA oxidation and stimulation of cellular uptake were indeed observed after transfection of 2/21 cells with cDNA coding for GGT. The mechanism of GGT-mediated AA oxidation was investigated in acellular systems, including GGT and its substrate glutathione. The process was observed in the presence of redox-active chelated iron(II) and of transferrin or ferritin, i.e., two physiological iron sources. Thus, membrane GGT activity-often expressed at high levels in human malignancies-can oxidize extracellular AA and promote its uptake efficiently.     

Suppression of platelet-activating factor generation and modulation of arachidonate metabolism by dietary enrichment with (n-9) eicosatrienoic acid or docosahexaenoic acid in mouse peritoneal cells

Several studies have shown that dietary n-3 polyunsaturated fatty acids (PUFAs) suppress platelet-activating factor (PAF) generation in leukocytes of humans and rodents, which is associated with the antagonism of arachidonic acid metabolism. Dietary eicosatrienoic acid (20:3n-9, ETrA) is also suggested to antagonize arachidonic acid (AA) metabolism, but its effect on PAF generation in leukocytes has not been defined. In the present study, we investigated the effects of an ETrA-rich diet on PAF generation and AA metabolism in mouse peritoneal cells, which were compared with those of a docosahexaenoic acid (DHA)-rich diet. Mice were fed a diet supplemented with a lipid preparation rich in ETrA, a DHA-rich fish oil (FO) or palm oil (PO) for 3 weeks, and peritoneal cells containing more than 80% of monocytes/macrophages were obtained. The peritoneal cells in the DHA and ETrA diet groups generated upon zymosan stimulation a smaller amount of PAF than cells in the PO diet group. In the peritoneal cells of the DHA diet group, AA contents in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were significantly lower than those in cells of the PO diet group, but those in phosphatidylinositol (PI) were not significantly different between the two dietary groups. A considerable amount of ETrA was incorporated into the peritoneal cells of the ETrA diet group, and AA was reduced as compared with the PO diet group. These changes occurred preferentially in PI but to a less extent in PC and PE. The amount of free AA released by the peritoneal cells upon zymosan stimulation was significantly reduced in the DHA diet group as compared with that in the PO diet group, whereas AA release was similar between the PO and ETrA diet groups. In conclusion, the effects of dietary ETrA on AA content in the phospholipid subclasses and AA release were quite different from those of dietary DHA, although both diets suppressed PAF generation in mouse peritoneal cells to a similar extent.     

Stories about acyl chains

The effect of dietary polyunsaturated fatty acids and alpha-tocopherol supplementation on erythrocyte lipid peroxidation and immunocompetent cells in mice was studied comparatively using seven dietary oils (15% oil/diet, w/w) including fish oil rich in eicosapentaenoic acid (EPA, 20:5, n-3) and docosahexaenoic acid (DHA, 22:6, n-3). A 43% increase in spleen weight, about twice as many spleen cells and no change in the subpopulations of spleen cells, as well as a significant depression of mitogen-induced blastogenesis of both T and B cells in the spleen were observed in mice fed fish oil for 30 days in comparison with soybean oil diet-fed mice. In the fish oil diet-fed mice, membranous lipid hydroperoxide (hydroperoxides of phosphatidylcholine and phosphatidylethanolamine) accumulation as a marker of oxidative senescence in red blood cells (RBC) was 2.7-3.5 times higher than that in mice fed soybean oil, although there was no difference in the plasma phosphatidylcholine hydroperoxide concentration. In spite of the supplementation of alpha-tocopherol to up to 10 times the level in the basal diet, the degeneration of spleen cells and the stimulated oxidative senescence of RBC found by the fish oil feeding could not be prevented. The results suggest that oral intake of excess polyunsaturated fatty acids, i.e. EPA and DHA, in a fish oil diet can lead to acceleration of membrane lipid peroxidation resulting in RBC senescence linked to the lowering of immune response of spleen cells, and that supplementation of alpha-tocopherol as antioxidant does not always effectively prevent such oxidative degeneration as observed in spleen cells and RBC in vivo.     

Transmission of symbiotic algae through sexual reproduction in hydra: movement of algae into the oocyte

The histological pathway by which intracellular symbiotic Chlorella move into the developing oocytes of hydra was investigated at the ultrastructural level. Algae move from within the digestive cells of the endoderm to within the oocytes of the ectoderm in a three-step process. First, the algae are released by digestive cells into the mesolamella (basement membrane). Second, the algae move as individual cells into the adjacent intercellular spaces of the ectoderm. Third, they are taken up by the oocyte by phagocytosis. This transfer occurs only in the central regions of the ovary, and only after oocytes have reached an advanced stage. Normally the mesolamella is separated from the ectodermal interstitial spaces by a layer of epitheliomuscular cell muscle processes. This layer degenerates in the region where algae will move into the ectoderm. This study shows that algae move as individual cells and not intracellularly within processes of the epithelial cells.     

产二十二碳六烯酸等多不饱和脂肪酸真菌的筛选*

从土壤中筛选出一株产二十二碳六烯酸（DHA）的丝状真菌,菌丝含油21.23％,DHA占总脂肪酸2.51％;同时含二十碳五烯酸（EPA）,占总脂肪酸的0．41％;不饱和脂肪酸占总脂肪酸的80％。经鉴定为头孢霉属（Caphalosporiumsp．）真菌。同时发现两株菌含EPA,经鉴定为小克银汉霉（Cunninghamellasp.）和毛霉（Mucorsp．）。在这几个属中发现DHA和EPA尚属首次。头孢霉菌DHA产量及百分含量和斜面菌种在不同温度下储藏有关。菌种在20℃储藏10天,在液体PDA培养基上发酵,DHA可占总脂肪酸11．27％,产量达63．35mg／L。     

微生物发酵产二十二碳六烯酸代谢机理的研究进展

二十二碳六烯酸(简称DHA)是一种重要的长链多不饱和脂肪酸,对人体具有重要的生理功能。微生物发酵生产的DHA与鱼油来源的DHA相比,具有诸多优点,其发展前景广阔。以下从发酵菌株、代谢途径、关键酶以及油脂积累机制等方面进行了综述,为通过代谢工程等技术手段进一步提高DHA产量提供了参考。     

Dihydroxyacetone induced autophagy in African trypanosomes

Dihydroxyacetone (DHA) was examined to explore its trypanocidal activity. The compound is easily taken up by trypanosomes via its aquaglyceroporins but is not converted to a glycolytic intermediate due to the lack of a respective kinase. Investigating the DHA-induced cell death it became evident that parasites die by autophagy rather than by necrosis or apoptosis. Since autophagy is not well studied in African trypanosomes our work offers a way to investigate the importance of autophagy for trypanosomes not only for stress coping but also for organelle reconstruction during differentiation.     

Fish oil diet affects on oxidative senescence of red blood cells linked to degeneration of spleen cells in mice

The effect of dietary polyunsaturated fatty acids and α-tocopherol supplementation on erythrocyte lipid peroxidation and immunocompetent cells in mice was studied comparatively using seven dietary oils (15% oil/diet, w/w) including fish oil rich in eicosapentaenoic acid (EPA, 20:5, n–3) and docosahexaenoic acid (DHA, 22:6, n–3). A 43% increase in spleen weight, about twice as many spleen cells and no change in the subpopulations of spleen cells, as well as a significant depression of mitogen-induced blastogenesis of both T and B cells in the spleen were observed in mice fed fish oil for 30 days in comparison with soybean oil diet-fed mice. In the fish oil diet-fed mice, membranous lipid hydroperoxide (hydroperoxides of phosphatidylcholine and phosphatidylethanolamine) accumulation as a marker of oxidative senescence in red blood cells (RBC) was 2.7–3.5 times higher than that in mice fed soybean oil, although there was no difference in the plasma phosphatidylcholine hydroperoxide concentration. In spite of the supplementation of α-tocopherol to up to 10 times the level in the basal diet, the degeneration of spleen cells and the stimulated oxidative senescence of RBC found by the fish oil feeding could not be prevented. The results suggest that oral intake of excess polyunsaturated fatty acids, i.e. EPA and DHA, in a fish oil diet can lead to acceleration of membrane lipid peroxidation resulting in RBC senescence linked to the lowering of immune response of spleen cells, and that supplementation of α-tocopherol as antioxidant does not always effectively prevent such oxidative degeneration as observed in spleen cells and RBC in vivo.     

Symbiotic association in Chlorella culture

Chlorella sorokiniana IAM C-212 has long been maintained in slant culture as a mixed strain, representing an associated natural microbial consortium. In this study, the consortium was separated and five nonalgal constituents, a fungal strain (CSSF-1), and four bacterial strains (CSSB-1, CSSB-2, CSSB-3, and CSSB-4) were isolated and identified. 16S rDNA sequence analysis revealed that strains CSSB-1, CSSB-2, CSSB-3, and CSSB-4 were close to Ralstonia pickettii (99.8% identity), Sphingomonas sp. DD38 (99.4% identity), Microbacterium trichotecenolyticum (98.6% identity), and Micrococcus luteus (98.6% identity) respectively. 18S rDNA sequence analysis revealed that strain CSSF-1 resembled Acremonium-like hyphomycete KR21-2 (98.8%). The fungal strain CSSF-1 and one of the bacterial strains, CSSB-3, were found to promote the growth of Chlorella while the presence of bacterial strains CSSB-1 and CSSB-2 had no effect. Strain CSSB-4 could not be subcultured so its role was not elucidated. These results show that the interaction between Chlorella and its symbionts under photoautotrophic conditions involved both mutualism and commensalisms. The chlorophyll content of mixed strain was stable in long-term cultivation (7 months) while the chlorophyll content of a pure culture showed a marked decline. Electron microscopic analysis showed the two bacterial strains CSSB-2 and CSSB-3 were harbored on the sheath excreted by Chlorella, while the fungal strain CSSF-1 and the bacterial strain CSSB-1 directly adhered to the Chlorella cell surface. This report is the first observation of a symbiotic relationship among fungus, bacteria, and Chlorella, and the first observation of direct adhesion of fungus and bacteria to Chlorella in a consortium.