{Omega}-3 and {omega}-6 polyunsaturated fatty acids block HERG channels

Dietary polyunsaturated fatty acids (PUFAs) have been reported to exhibit antiarrhythmic properties, which have been attributed to their availability to modulate Na⁺, Ca²⁺, and several K⁺ channels. However, their effects on human ether-a-go-go-related gene (HERG) channels are unknown. In this study we have analyzed the effects of arachidonic acid (AA, -6) and docosahexaenoic acid (DHA, -3) on HERG channels stably expressed in Chinese hamster ovary cells by using the whole cell patch-clamp technique. At 10 µM, AA and DHA blocked HERG channels, at the end of 5-s pulses to –10 mV, to a similar extent (37.7 ± 2.4% vs. 50.2 ± 8.1%, n = 7–10, P > 0.05). 5,6,11,14-Eicosatetrayenoic acid, a nonmetabolizable AA analog, induced effects similar to those of AA on HERG current. Both PUFAs shifted the midpoint of activation curves of HERG channels by –5.1 ± 1.8 mV (n = 10, P < 0.05) and –11.2 ± 1.1 mV (n = 7, P < 0.01). Also, AA and DHA shifted the midpoint of inactivation curves by +12.0 ± 3.9 mV (n = 4; P < 0.05) and +15.8 ± 4.3 mV (n = 4; P < 0.05), respectively. DHA and AA accelerated the deactivation kinetics and slowed the inactivation kinetics at potentials positive to +40 mV. Block induced by DHA, but not that produced by AA, was higher when measured after applying a pulse to –120 mV (IO). Finally, both AA and DHA induced a use-dependent inhibition of HERG channels. In summary, block induced by AA and DHA was time, voltage, and use dependent. The results obtained suggest that both PUFAs bind preferentially to the open state of the channel, although an interaction with inactivated HERG channels cannot be ruled out for AA. K⁺ channel; membrane currents; ion channels; arrhythmia; antiarrhythmics     

Ontogenetic vertical distribution and diel migration of the copepod Eucalanus inermis in the oxygen minimum zone off northern Chile (20-21{degrees} S)

The vertical and ontogenetic distribution, and diel verticalmigration (DVM), of Eucalanus inermis in relation to the strongvertical gradient in oxygen concentration associated with anintense oxygen minimum zone (OMZ) were studied at a coastalarea off northern Chile (20–21° S). A close relationshipbetween the abundance of the whole copepod population and lowoxygen waters was found, with most developmental stages remainingnear the base of the oxycline (30–80 m) and within theupper zone of the OMZ (30–200 m). All stages performedDVM but not at all the stations, mainly between the 30–60and 60–200 m strata; a small fraction (<20%) appearedin the surface layer (0–30 m) mostly at night. This strategyof movement would result in a better utilization of food resourcessince the strong physical and chemical gradients at the baseof the oxycline and upper OMZ boundary might serve as a siteof particle accumulation. A secondary fluorescence peak was,in fact, found at all the stations, coinciding with minimaldissolved oxygen (DO, <1 mL O₂ L^–1) at the base ofthe oxycline or in the upper OMZ boundary. The relevance ofthe biogeochemical flux involved in this diel migration patternwas assessed by calculating the potential active input of carbonand nitrogen from the upper layers into deeper the OMZ.     

The Enzymic Decarboxylation of {gamma}-methyleneglutamic Acid by Plant Extracts

-Methyleneglutamic acid, an acidic amino-acid isolated fromgroundnut plants, was decarboxylated by enzymes present in extractsof Capsicum fruits, barley roots, and tulip leaves, and alsoby intact cells of Clostridium welchii S.R. I2. The amino-acidwas attacked in a similar manner to, but in all cases at a slowerrate than, l-glutamic acid. The nature of the enzyme responsiblefor the decarboxylation of -methyleneglutamic acid was furtherinvestigated using preparations from barley roots (which donot contain the amino-acid) and from tulip leaves (in whichthe amino-acid is normally present, together with larger amountsof its amide form, -methyleneglutamine). The effects of pH,inhibitors, and partial heat denaturation upon the enzyme systemspresent in the barley and tulip extracts indicated that a singleenzyme was responsible for the decarboxylation of both l-glutamicacid and -methyleneglutamic acid. Although the Cl. welchii rapidlydeamidated and then decarboxylated l-glutamine, -methyleneglutaminewas not attacked by the organism.     

Novel Asn-linked oligosaccharides terminating in GaINAc{beta}(1->4)[Fuca(1->3)]GlcNAcB(1->{bullet}) are present in recombinant human Protein C expressed in human kidney 293 cells

Recombinant human Protein C (rHPC), expressed in human kidney293 cells, has a higher anticoagulant activity than plasma HPC,while its in vivo circulatory half-life is essentially unalteredcompared to that of the natural protein. In seeking to elucidatethe molecular basis for the improved efficacy of the recombinantantithrombotic drug, we focused on the carbohydrate moiety ofrHPC. Protein C is a heavily post-translationally modified serineprotease with four N-glycosylation sites. Glycosyl compositionanalysis of rHPC revealed a 5-fold higher fucose content anda 2-fold lower sialic acid content compared to plasma HPC. Inaddition, we found that rHPC contains N-acetylgaiac-tosamine(2.6 mol GalNAc/mol rHPC) in its Asn-linked oligosaccharides,while plasma HPC is devoid of GalNAc. The Asn-linked oligosaccharidesof rHPC were released by N-glycanase and separated into 25 fractionsby high-pH anion-exchange chromatography. The most abundantoligosaccharides were structurally characterized by glycosylcomposition and linkage analysis, in conjunction with ¹H-NMRspectroscopy at 600 MHz. The structure of the major neutraloligosaccharide in rHPC was determined to be: Two representatives of the sialylated oligosaccharides in rHPCare: and Thus, many of the Asn-linked oligosaccharides in rHPC were foundto terminate in GaINAcß(1     

Part of the Lysyl Residues in Wheat {alpha}-Amylase is Methylated as N-{varepsilon}-Trimethyl Lysine

Amino acid analyses of wheat -amylase purified from germinatingseeds by affinity chromatography showed a high content of amodified lysyl residue. The modified residue was identifiedas N--trimethyl lysine. The presence of trimethyl lysine in-amylase is discussed in terms of isozymes. ¹ Present address: National Institutes of Health, Bldg. 10,Rm. 9B-15, Bethesda, MD 20205, U.S.A. (Received August 20, 1981; Accepted March 19, 1982)     

Distribution of Diacylglycerylhydroxymethyltrimethyl-{beta}-alanine (DGTA) and Phosphatidylcholine in Brown Algae

Lipids were analyzed in thirteen species of brown algae collectedat the seashore near Tokyo, Japan. Diacylglycerylhydroxymethyltrimethyl-ß-alanine(DGTA), a recently identified betaine lipid, was found as amajor lipid component in eight species of brown algae examined,namely, Ishige okamurai, Dictyota dichotoma, Pachydictyon coriaceum,Padina arborescens, Hizikia fusiformis, Sargassum horneri, S.ringgoldianum and S. thunbergii. However, phosphatidylcholine(PC) was not detected in any of these algae except I. okamurai.By contrast, PC was found as a major lipid component in fiveother species, namely, Colpomenia sinuosa, Endarachne binghamiae,Scytosiphon lomentarius, Eisenia bicyclis, Undaria pinnatifida.These algae in turn did not contain detectable amounts of DGTA.The fatty acid composition of four selected species, S. lomentarius,U. pinnatifida, D. dichotoma and H. fusiformis, was also studied.The fatty acid components of DGTA in D. dichotoma and H. fusiformiswere similar to those of PC in U. pinnatifida, the major componentsbeing 16:0, 18:2 and 20:46 (also 16:1 in D. dichotoma). (Received December 14, 1990; Accepted April 10, 1991)     

Human melanoma and Chinese hamster ovary cells galactosylate n-alkyl-{beta}-glucosides using UDP gal:GlcNAc {beta}1,4 galactosyltransferase

We previously showed that human melanoma, CHO and other cellscan convert ß-xylosides into structural analogs ofganglioside G_M3. We have investigated several potential acceptorsincluding a series of n-alkyl-ß-D-glucosides (n =6–9). All were labeled with ³H-galactose when incubatedwith human melanoma cells. Octyl-ß-D-glucoside (GlcßOctyl)was the best acceptor, whereas neither octyl--D-glucoside norN-octanoyl-methylglucamine (MEGA 8) were labeled. Analysis ofthe products by a combination of chromatographic methods andspecific enzyme digestions showed that the acceptors first receiveda single Galß1,4 residue followed by an 2,3 linkedsialic acid. Synthesis of these products did not affect cellviability, adherence, protein biosynthesis, or incorporationof radio-labeled precursors into glycoprotein, glycolipid orproteoglycans. To determine which ß1,4 galactosyltransferase synthesized Galß1,4GlcßOctyl,we analyzed similar incubations using CHO cells and a mutantCHO line (CHO 761) which lacks GAG-core specific ß1,4galactosyltransferase. The mutant cells showed the same levelof incorporation as the control, eliminating this enzyme asa candidate. Thermal inactivation kinetics using melanoma cellmicrosomes and rat liver Golgi to galactosylate GlcßOctylshowed the same half-life as UDP-Gal:GlcNAc ß1,4 galactosyltransferase,whereas LacCer synthase was inactivated at a much faster rate.We show that GlcßOctyl is a substrate for purifiedbovine milk UDP-Gal:GlcNAc ß1,4 galactosyltransferaseFurthermore, the galactosylation of GlcßOctyl by CHOcell microsomes can be competitively inhibited by GlcNAc orGlcNAcßMU . These results indicate that UDP-Gal:GlcNAcß1,4 galactosyltransferase is the enzyme used forthe synthesis of the alkyl lactosides when cells or rat liverGolgi are incubated with alkyl ß glucosides. alkylglucosides galactosyltransferase glycolipid artificial acceptors     

Developmental Variation of the Neurotoxin, {beta}-N-Oxalyl-L-{alpha},{beta}-diamino propionic acid (ODAP), in Lathyrus sativus

Several species of the tribe Viceae (Leguminosae) produce non-proteinamino acids that are toxic to man and animals. The neurotoxinß-N-oxalyl-L-,ß-diamino propionic acid (ODAP)in cultivated Lathyrus sativus causes human neurolathyrism,a neurological disease resulting in the paralysis of lower limbs.Surveys have shown that there are large scale variations betweenspecies of Lathyrus and varieties of L. sativus for the amountof cellular ODAP. In the present investigation, thin-layer chromatographyand chemical analysis were used to study developmental variationin the amount of ODAP in tissues and organs of L. sativus. Theresults confirmed that the rate of synthesis and accumulationof ODAP varied during plant development. Increased rates ofsynthesis were confirmed in young seedlings and in the developingfruits of L. sativus.Copyright 1994, 1999 Academic Press Lathyrus sativus, neurotoxin, ODAP, plant development     

Effects of Fluorogibberellins on Plant Growth and Gibberellin 3r{beta}-Hydroxylases

3rß-Fluorogibberellin A₉ (3rß-fluoro-GA₉),3rßfluoro-GA₂₀, 3rß-fluorodeoxygibberellinC (3rß-fluoro-DGC) and 13-fluoro-GA₉ were prepared,and their effects on plant growth and gibberellin (GA) 3rß-hydroxyIaseswere examined. 3rß-Fluoro-GA₉ and 3rß-fluoro-GA₂₀promoted the growth of dwarf rice (Oryza sativa L. cv. Tan-ginbozu)seedlings to three times higher than the control seedlings ata dosage of 3 µ plant^–1, and 3rßfluoro-DGCto twice higher at the same dosage. 3rßg-Fluoro-GA₉was active in cucumber (Cucumis sativus L.) hypocotyl assay,its activity being about one-thirtieth as much as that of GA₄.3rß-Fluoro-GAs were active per se in promoting theshoot elongation of rice. 3rß-Fluoro-DGC inhibitedthe 3rß-hydroxylation of [³H₂]GA₉ to [³H]GA₄ by GArß-hydroxylase from bean (Phaseolus vulgaris L.),but 3rß-fluoro-GA₉ and 3rß-fluoro-GA₂₀ didnot show any effects on the enzyme activity. These 3rß-fluoro-GAsalso showed no or only a weak inhibitory effect on the rß-hydroxylasefrom pumpkin (Cucurbita maxima L.). 13-Fluoro-GA₉ promoted growthof rice and cucumber seedlings, and inhibited the 3rß-hydroxylasesfrom both bean and cucumber. 13-Fluoro-GA₉was converted into13-fluoro-GA₄ and 2,3-didehydro-13-fluoro-GA₉, in a cell-freesystem from bean, and conversion of 13-fluoro-GA₉ into 13-fluoro-GA₄was also observed in a cell-free system from pumpkin. Theseresults suggest that 13-fluoro-GA₉ is one of the substratesof GA 3rß-hydroxy-lases, and that 13-fluoro-GA₉ isactive as a result of the conversion to 13-fluoro-GA₄ in riceand cucumber seedlings. (Received October 27, 1997; Accepted March 13, 1998)     

Modification of sialic acids by 9-O-acetylation is detected in human leucocytes using the lectin property of influenza C virus

Influenza C virus spike glycoprotein HEF specifically recognizesglycoconjugates containing 9-O-acetyl-N-acetylneuraminic acid.The same protein also contains an esterase activity. Takingadvantage of these two properties, influenza C virus was usedas a very sensitive probe for the detection of traces of 9-O-acetyl-N-acetylneuraminicacid in human leucocytes. The binding of influenza C virus toleucocyte glycoproteins and gangliosides separated by sodiumdodecyl sulphate–polyacrylamide gel electrophoresis andthin-layer chromatography, respectively, was assayed using achromogenic esterase substrate. In this way, glycoproteins ofB-lymphocytes and T-lymphocytes were found to contain 9-O-acetylatedsialic acids. Of the various 9-O-acetylated gangliosides detected,one had the characteristics of 9-O-acetylated G^D3. The identificationof 9-O-acetylated sialic acids on distinct glycoproteins andglycolipids should be helpful in assigning a physiological roleto this sugar. O-acetylation gangliosides influenza C virus lymphocytes sialic acids     

TFF3 modulates NF-{kappa}B and a novel negative regulatory molecule of NF-{kappa}B in intestinal epithelial cells via a mechanism distinct from TNF-{alpha}

Trefoil factor 3 (intestinal trefoil factor) is a cytoprotective factor in the gut. Herein we compared the effect of trefoil factor 3 with tumor necrosis factor- on 1) activation of NF-B in intestinal epithelial cells; 2) expression of Twist protein (a molecule essential for downregulation of nuclear factor-B activity in vivo); and 3) production of interleukin-8. We showed that Twist protein is constitutively expressed in intestinal epithelial cells. Tumor necrosis factor- induced persistent degradation of Twist protein in intestinal epithelial cells via a signaling pathway linked to proteasome, which was associated with prolonged activation of NF-B. In contrast to tumor necrosis factor, trefoil factor 3 triggered transient activation of NF-B and prolonged upregulation of Twist protein in intestinal epithelial cells via an ERK kinase-mediated pathway. Unlike tumor necrosis factor-, transient activation of NF-B by trefoil factor 3 is not associated with induction of IL-8 in cells. To examine the role of Twist protein in intestinal epithelial cells, we silenced the Twist expression by siRNA. Our data showed that trefoil factor 3 induced interleukin-8 production after silencing Twist in intestinal epithelial cells. Together, these observations indicated that 1) trefoil factor 3 triggers a diverse signal from tumor necrosis factor- on the activation of NF-B and its associated molecules in intestinal epithelial cells; and 2) trefoil factor 3-induced Twist protein plays an important role in the modulation of inflammatory cytokine production in intestinal epithelial cells. trefoil factor 3; signal transduction     

Column Chromatographic Separation of Chlorophyllide {alpha} and Pheophorbide {alpha}

Chlorophyllide a, pheophorbide a, chlorophyll a and pheophytina were separated in a short time by anion-exchange chromatographywith a short column of DEAE-Sepharose CL-6B. (Received February 16, 1984; Accepted April 13, 1984)     

Expression of {beta}-galactoside {alpha}2,6 sialyltransferase blocks synthesis of polysialic acid in Xenopus embryos

Polysialic acid is a developmentally regulated carbohydratestructure found on neural cell adhesion molecules (NCAM). Expressionof ß-galactoside 2,6-sialyltransferase in Xenopusembryos, by injection of mRNA, prevents the polysialylationof NCAM, presumably by introducing a different type of sugarlinkage that terminates chain elongation. Abnormalities in neuraldevelopment result from this treatment, but in general the bodyplan of the injected embryos is not severely affected. The resultsprovide evidence that the mis-expression of glycosyltransferasescan be used to interfere with the normal pattern of glycosylationin whole organisms. glycosylation NCAM polysialic acid Xenopus development     

Transcription of the {beta}-galactoside {alpha}2,6-sialyltransferase gene in B lymphocytes is directed by a separate and distinct promoter{dagger}

A single human gene, SIAT1, encodes the ß-galactoside     

Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human {beta}1,2-N-acetylglucosaminyltransferase I

Spodoptera frugiperda (Sf9)-cells differ markedly in their proteinglycosylation capacities from vertebrate cells in that theyare not able to generate complex type oligosaccharide side chains.In order to improve the oligosaccha ride processing propertiesof these cells we have used baculovirus vectors for expressionof human (ß1,2-N-acetylglucosaminyltransferase I (hGNT-I),the enzyme catalysing the crucial step in the pathway leadingto complex type N-glycans in vertebrate cells. One vector (Bac/GNT)was designed to express unmodified GNT-I protein, the secondvector (Bac/tagGNT) to express GNT-I protein with a tag epitopefused to its N-terminus. In Sf9-cells infected with Bac/tagGNT-virusa protein of about 50 kDa representing hGNT-I was detected withan antiserum directed against the tag epitope. HGNT-I activitywas increased at least threefold in lysates of infected cellswhen N-acetylglucosamine (GlcNAc)-free ovalbumine was used assubstrate. To monitor hGNT-I activity in intact Sf9-cells, theglycosylation of coexpressed fowl plague virus hemagglutinin(HA) was investigated employing a galactosylation assay andchromatographic analysis of isolated HA N-glycans. Coexpressionof hGNT-I resulted in an at least fourfold increase of HA carryingterminal GlcNAc-residues. The only structure detectable in thisfraction was GlcNAcMan₃GlcNAc₂. These results show that hGNT-Iis functionally active in Sf9-cells and that the N-glycans ofproteins expressed in the baculovirus/insect cell system areelongated by coexpression of glycosyltransferases of vertebrateorigin. Complete complex type oligosaccharide side chains werenot observed when hGNT-I was overexpressed, thus supportingthe concept that Sf9-cells do not contain glycosyltransferasesacting after hGNT-I. ß1,2-N-acetylglucosaminyltransferase I baculovirus expression of recombinant protiens N-glycosylation in Sf9-cells     

Nature of light-induced absorbance changes at 682 m{micro} and 702 m{micro} in photosynthesis of Anacystis nidulans

Light-induced absorbance changes in the region around the redabsorption band of chlorophyll a were measured in cells andlamella fragments of Anacystis nidulans. In both materials,absorbance decreases were observed at 702 mµ and 682 mµ.(The pigments are designated as P₇₀₀ and P₆₈₀.) The nature ofP₆₈₀ was investigated with special reference to its relationshipto P₇₀₀. In the cells, light absorbed by chlorophyll a causedan absorbance decrease at 682 mµ; Simultaneous illuminationwith light absorbed by phycocyanin caused a partial recoveryof the absorbance decrease. Similar results were observed withthe light-induced absorbance change at 702 mµ. This indicatesthat P₆₈₀ is also an electron carrier in the electron transportchain and occupies a place between the two photoreactions. Inlamella fragments, both the light-induced reversible absorbancechanges of P₆₈₀ and P₇₀₀ appeared in the presence of an electrondonor system; i.e., ascorbate and 2,6-dichlorophenolindophenolor N,N,N',N'-tetramethyl-l,4-phenylenediamine. The experimentsin which the oxidation-reduction potential of the reaction mediumwas changed showed that both P₆₈₀ and P₇₀₀ are one-electroncarriers, having a normal oxidation-reduction potential of 0.44v (assuming that the normal oxidation-reduction potential ofthe ferricyanide-ferrocyanide system is 0.409 v). A possibilitywas suggested that the absorbance change observed at 682 mµis another expression of the oxidation-reduction reaction ofP₇₀₀). (Received October 30, 1968; )