DEAE-Sephadex Column Research Articles

Purification of wild type and recombinant xylanases and evaluation of their arabinoxylan hydrolysis ability

Arabinoxylan hydrolyzing enzymes are very rich and diversified groups, among these, endo-1,4-β-xylanase is the most important group which catalyzes the cleavage of xylan main skeleton to release arabinoxylan oligosaccharides such as D-xylose, xylobiose, L-arabinose, xylotetraose, xylopentose. This group of enzyme was efficiently produced from natural microorganism including bacteria, yeast and fungi and recombinant strains. In this study, we have purified wild-type and recombinant xylanase to compare the difference in hydrolysis products of these two enzymes. Using sephadex G-100 and DEAE-sephadex column chromatography, we have purified xylanase from wild-type Aspergillus niger DSM1957 with molecular weight of 25 kDa, specific activity of 3240 IU/mL, the purity increases 1.68 folds compare to crude extract, and a recovery rate of 36%. The recombinant enzyme from Pichia pastoris GS115/pPXlnA was purified by ProbondTM affinity chromatography column, with a molecular weight of 36 kDa; the purity increased 3.2 folds than the crude extract, and with the recovery efficiency of 21.1%. The activity of commercial wild-type xylanase was better than that of our recombinant and wild-type enzymes. Our wild-type xylanase could hydrolyze arabinoxylan into L-arabinose in 50 mM CH3COONa pH 5.0. Our results showed that xylanases purified from different sources were stable and highly specific to xylan. Xylanase has potential application in the production of high quality biotechnological products.

Production, immobilization and application of invertase from new wild strain Cunninghamella echinulata PA3S12MM.

The objective of this study was to determine the best conditions to produce invertase by Cunninghamella echinulata PA3S12MM and to immobilize and apply the enzyme. The maximum production was verified in 8days of cultivation at 28°C supplemented with 10gL-1 apple peel, reaching 1054.85Uml-1 . The invertase was purified from the DEAE-Sephadex column. The derivative immobilized in alginate-gelatin-calcium phosphate showed reusability >50% for 19 cycles. The derivative immobilized in glutaraldehyde-chitosan showed greater thermostability and at a different pH. The hydrolysis of 15ml of sucrose 500gL-1 in a fixed bed reactor (total volume of 31ml) produced 24.44µmolmin-1 of glucose and fructose at a residence time of 30min and a conversion factor of 0.5. The new wild strain C. echinulata PA3S12MM presents high invertase production in medium supplemented with an agro-industrial residue and the immobilized enzyme showed high thermal stability and resistance at a different pH. The fungus C. echinulata PA3S12MM is an excellent producer of invertases in Vogel medium supplemented with apple peel. The enzyme is promising for industrial application since it has good performance in reusability and inverted sugar production.

Purification, characterization, and immunomodulatory activity of a lectin from the seeds of horse chestnut (Aesculus hippocastanum L.)

This work aimed to purify, characterize, and evaluate the immunomodulatory activity of a lectin from Aesculus hippocastanum seeds (AhSL). The lectin was isolated from saline extract through chromatography on a DEAE-Sephadex column. Protein homogeneity and subunit composition were evaluated with polyacrylamide gel electrophoresis (PAGE) under native and denaturing (SDS-PAGE) conditions, respectively. AhSL was evaluated for trypsin inhibitor activity, carbohydrate-binding specificity, and the effects of cations (Ca2+, Mn2+, and Mg2+), temperature, and pH on its hemagglutinating activity. Lectin stability under heating and pH variation was evaluated through fluorometric analysis. The activity of this lectin (12.5 µg/mL) in modulating cytokine release and nitric oxide production by mouse splenocytes was assessed. Isolated AhSL (purification fold: 68.8) showed a single polypeptide band in PAGE for acidic proteins and two polypeptide bands of approximately 68 and 124 kDa in SDS-PAGE. Its hemagglutinating activity was inhibited by monosaccharides, disaccharides, and glycoproteins, but was not affected by cations, and remained stable after heating (30–100 °C) and under pH 4.0–8.0. Fluorometric analysis revealed that the overall lectin conformation was not altered after heating to 100 °C and remained stable under various pH values. AhSL also showed trypsin inhibitor activity (specific activity: 1,280.31 U/mg) and induced the release of interleukins 10 and 6, tumor necrosis factor α, and nitric oxide by splenocytes. In conclusion, AhSL is a bioactive protein with promising biotechnological potential, which may find applications as immunological targeting or antitumor agents.

Novel Thermotolerant Amylase from Bacillus licheniformis Strain LB04: Purification, Characterization and Agar-Agarose.

This study analyzed the thermostability and effect of calcium ions on the enzymatic activity of α-amylase produced by Bacillus licheniformis strain LB04 isolated from Espinazo Hot springs in Nuevo Leon, Mexico. The enzyme was immobilized by entrapment on agar-agarose beads, with an entrapment yield of 19.9%. The identification of the bacteria was carried out using 16s rDNA sequencing. The enzyme was purified through ion exchange chromatography (IEX) in a DEAE-Sephadex column, revealing a protein with a molecular weight of ≈130 kDa. The enzyme was stable at pH 3.0 and heat stable up to 80 °C. However, the optimum conditions were reached at 65 °C and pH 3.0, with a specific activity of 1851.7 U mg−1 ± 1.3. The agar-agarose immobilized α-amylase had a hydrolytic activity nearly 25% higher when compared to the free enzyme. This study provides critical information for the understanding of the enzymatic profile of B. licheniformis strain LB04 and the potential application of the microorganisms at an industrial level, specifically in the food industry.

Polysaccharide IV from Lycium barbarum L. Improves Lipid Profiles of Gestational Diabetes Mellitus of Pregnancy by Upregulating ABCA1 and Downregulating Sterol Regulatory Element-Binding Transcription 1 via miR-33.

Lycium barbarum L. (LBL) has beneficial effects on gestational diabetes mellitus (GDM) but the related mechanism remains unclear. Polysaccharides of LBL (LBLP) are the main bioactive components of LBL. miR-33, ATP-binding cassette transporter A1 (ABCA1) and sterol regulatory element-binding transcription 1 (SREBF1) affect lipid profiles, which are associated with GDM risk. LBLP may exert protective against GDM by affecting these molecules. Four LBLP fractions: LBLP-I, LBLP-II, LBLP-III, and LBLP-IV were isolated from LBL and further purified by using DEAE-Sephadex column. The effects of purified each fraction on pancreatic beta cells were comparatively evaluated. A total of 158 GDM patients were recruited and randomly divided into LBL group (LG) and placebo group (CG). miR-33 levels, lipid profiles, insulin resistance and secretory functions were measured. The association between serum miR-33 levels and lipid profiles were evaluated by using Spearman’s rank-order correlation test. After 4-week therapy, LBL reduced miR-33 level, insulin resistance and increased insulin secretion of GDM patients. LBL increased the levels of ABCA1, high-density lipoprotein cholesterol (HDL-C) and reduced miR-33, SREBF1, low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), triglyceride (TG), and malondialdehyde. Homeostatic model assessment of β-cell function and insulin resistance was lower in LG than in CG, whereas homeostatic model assessment of β-cell function and insulin secretory function was higher in LG than in CG. There was a strong positive association between miR-33 level and TG, or TC and or LDL-C, and a strong negative association between miR-33 level and HDL-C. The levels of miR-33 had negative relation with ABCA1 and positive relation with SREBF1. ABCA1 has negative relation with TG, TC, and LDL-C and positive relation with HDL-C. Inversely, SREBF1 had positive relation with TG, TC, and LDL-C and negative relation with HDL-C. The main bioactive compound LBLP-IV of LBL increased insulin secretion of beta cells and the levels of ABCA1, and reduced miR-33 levels and SREBF1 in beta cells. However, LBLP-IV could not change the levels of these molecules anymore when miR-33 was overexpressed or silenced. LBLP-IV had the similar effects with LBL on beta cells while other components had no such effects. Thus, LBLP-IV from LBL improves lipid profiles by upregulating ABCA1 and downregulating SREBF1 via miR-33.

Antihypertensive Effect of Peptides from Sesame, Almond, and Pumpkin Seeds: In-silico and In-vivo Evaluation

Plant proteins are important functional ingredients in many processed food products. In particular, globular proteins from various sources play an important role in many food products. In the current study, 11S globulin protein from white sesame seeds, amandin protein from almond seeds, and cucurbitin protein from pumpkin seeds were extracted by means of ammonium sulfate precipitation and purified by anion-exchange chromatography on a DEAE-Sephadex column (20 × 30 cm). Amandin protein of almond and cucurbitin protein of pumpkin seeds both belong to the 11S globulin family. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of protein samples treated with reducing agents revealed that the isolated 11S globulin from white sesame seeds was composed of an acidic (30－33 kDa) and basic (20－24 kDa) polypeptide, amandin from almond seeds was composed of an acidic (40－42 kDa) and basic (15－20 kDa) polypeptide, and cucurbitin from pumpkin seeds was composed of an acidic (35－42 kDa) and basic (20－25 kDa) polypeptide. The alpha amylase-inhibiting activities of the three proteins was determined. Cucurbitin of pumpkin seeds had a greater alpha amylase inhibitory effect (－86%) than 11S globulin of sesame (82.6%) and amandin protein of almond seeds (76%). The antihypertensive effect of the three proteins was evaluated by a chrioallantoin membrane assay in chick embryos, which revealed that cucurbitin protein showed higher vasodilatation activity than the other two proteins.

Alkaline serine proteases from Helicoverpa armigera: potential candidates for industrial applications.

We characterized trypsin- and chymotrypsin-like serine alkaline proteases from cotton bollworm, Helicoverpa armigera, for their probable potential application as additives in various bio-formulations. Purification was achieved by using hydroxylapatite, DEAE sephadex and CM sephadex columns, which resulted in increased enzyme activity by 13.76- and 14.05-fold for trypsin and chymotrypsin, respectively. Michaelis-Menten constants (Km ) for substrates of trypsin and chymotrypsin, BApNA and SAAPFpNA, were found to be 1.25 and 0.085 mM, correspondingly. Fluorescent zymogram analysis indicated the presence of five trypsin bands with molecular masses of ∼21, 25, 38, 40, and 66 kDa and two chymotrypsin bands with molecular masses of ∼29 and 34 kDa in SDS-PAGE. The optimum pH was 10.0 and optimum temperature was 50°C for proteolytic activity for the purified proteases. The proteases were inhibited by synthetic inhibitors such as PMSF, aprotonin, leupeptin, pefabloc, and antipain. TLCK and TPCK inhibited about 94 and 90% of trypsin and chymotrypsin activity, respectively, while EDTA, EGTA, E64, pepstatin, idoacetamide, and bestatin did not affect the enzymes. The purified enzymes exhibited high stability and compatibility with metal ions; oxidizing, reducing, and bleaching agents; organic solvents; and commercial detergents. Short life cycles, voracious feeding behavior, and production of multiple forms of proteases in the midgut with rapid catalytic activity and chemostability can serve H. armigera as an excellent alternative source of industrially important proteases for use as additives in stain removers, detergents, and other bio-formulations. Identification of enzymes with essential industrial properties from insect species could be a bioresource.

Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose

A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma.

A modified method for synthesis of [γ-32P] labelled adenosine triphosphate

Production of [γ-32P]-ATP using three glycolysis enzymatic reaction i.e. glyceraldehyde 3-phosphate dehydrogenase, 3-phosphoglyceric phosphokinase and lactate dehydrogenase has been conducted. dl-glyceraldehyde 3-phosphate, Adenosine Diphosphate and H 3 32 PO4 was used as precursors for this reaction. Purification of [γ-32P]-ATP was performed by using DEAE-Sephadex column chromatography. The result suggested that this simple method could be used for producing [γ-32P]-ATP to support the provision of radiolabeled nucleotide for biotechnology research in Indonesia.

Purification and characterisation of α-amylase produced by mutant strain of Aspergillus oryzae EMS-18

α-Amylase produced by a mutant strain of Aspergillus oryzae EMS-18 has been purified to homogeneity as judged by sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified by using 70% ammonium sulphate precipitation followed by anion exchange chromatography on DEAE-Sephadex column and gel filtration on Sephadex G-100. An enzyme purification factor of 9.5-fold was achieved with a final specific activity of 1987.7 U/mg protein and overall yield of 23.8%. The molecular weight of purified α-amylase was estimated to be 48 kDa by SDS-PAGE. The purified enzyme revealed an optimum assay temperature and pH 40°C and 5.0, respectively. Except Ca++ all other metal ions such as Mg, Mn, Na, Zn, Ni, Fe, Cu, Co and Ba were found to be inhibitory to enzyme activity.

Extracellular phytase from Aspergillus niger CFR 335: purification and characterization.

Phytase, that is extensively used as a feed additive is capable of hydrolyzing phytic acid, an antinutrient found in about 60-80% of all the plant commodities. This enzyme improves the bioavailability of essential minerals such as Ca(2+), Mg(2+), P, Zn(2+), Fe(3+), that are bound to phytic acid. An extracellular phytase from a local fungal isolate, Aspergillus niger CFR 335 was purified to homogeneity through a three-step column chromatography using DEAE-Sephadex anion exchanger. An active fraction of the enzyme was obtained with NaCl gradient of 2.5M in DEAE Sephadex column. The enzyme was purified up to 16 fold with a yield of 28.5%. Substrate specificity studies revealed a highest specific activity of 32.6 ± 3.1 U/mg for sodium phytate with the Km value of 0.08 ± 0.1mM. The molecular weight of the enzyme was 66kDa with an optimum temperature of 30°C and pH4.5. Up to 80% of the activity was retained even after storing the enzyme for 6months at 4°C.

Production of cellulase enzyme by Trichoderma reesei Cefl9 and its application in the production of bio-ethanol.

The present study aimed at the production of cellulase enzyme from the cellulolytic fungi Trichoderma reesei CEF19 and subsequent application of the cellulase for the fermentation of ethanol. For the same, the cellulolytic fungi, Trichoderma reesei CEF19 was isolated and was allowed to produce cellulase enzyme using optimized conditions. The cellulase enzyme was extracted and purified with the help of ammonium sulphate precipitation, dialysis followed by ion exchange chromatography with DEAE-Sephadex column. The purified cellulase enzyme was characterized using SDS-PAGE analysis. The saccharification of the cellulosic substrates was done using the cellulase enzyme. The fermentation of saccharified cellulosic substrates into ethanol was carried out using Saccharomyces cerevisiae. From the results obtained, rice straw was found to be the better source for the ethanol production when compared to the other substrates.

One-Step Chromatographic Purification of Helicobacter pylori Neutrophil-Activating Protein Expressed in Bacillus subtilis

Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of Helicobacter pylori (H. pylori), is capable of activating human neutrophils to produce reactive oxygen species (ROS) and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis). This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from B. subtilis in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection.

Binding Stoichiometry of a Recombinant Selenophosphate Synthetase with One Synonymic Substitution E197D to a Fluorescent Nucleotide Analog of ATP, TNP-ATP

The transformation of the strain DH5α TM-T1R with plasmid vector pET11a containing the cloned gene of bacterial selenophosphate synthetase (SPS), selD, from the E. coli BL21-Gold (DE3) strain gives an overproducing strain of SPS with one synonymic substitution, E197D. The transformation efficiency was estimated as 8 × 108 CFU/μg plasmid DNA. 28 mg of highly purified preparation of recombinant SPS capable of binding TNP-ATP was eluted from DEAE-Sephadex column in amount of 15 % from the total soluble protein in crude extract. The fluorescent derivative of ATP, 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine-5′-triphosphate (TNP-ATP), was used as a synthetic analog of the substrate for the monitoring and quantitative analysis of the functional activity of SPS. The non-linear regression analysis of the saturation curve of TNP-ATP binding to D197 SPS with GraphPad Prism software fits to a model with 2 distinct binding sites with KDs different in order. The SPS existence in a form of tetramer in given reaction conditions, in accordance with the concentration stoichiometry of 4 moles of TNP-ATP to 1 mole of recombinant protein, is being discussed. The tetramer structure was predicted with molecular modelling software YASARA and modelled in vacuum using steepest descent minimization energy method. We hypothesize here the recombinant SPS exists as a dimer in solution with two active sites capable of ATP binding in each subunit.

Kinetic and expression profiling of cytosolic pyruvate kinase enzyme during seed development of Indian mustard (Brassica juncea)

In the recent years tremendous progress has been achieved in deconstructing the oil biosynthetic pathways, majority of which is in Arabidopsis. Glycolysis is fundamental to this process as it is the cardinal supplier of precursors for fatty acid metabolism. Recent reports suggest that modification and expression of pyruvate kinase (PK), a crucial regulatory enzyme involved in glycolysis is one of the plausible ways to alter seed oil content in plants. In the present study we evaluated the kinetic behavior and expression profiling of pyruvate kinase, associated with seed development in a major oilseed crop B. juncea. Developmental profiling of the enzyme showed that enzyme activity was highest during middle stage (35 DAF) of seed development which is strongly corroborated by the expression profiling of the enzyme using RT-PCR approach. Oil accumulation pattern also correlated with the enzyme expression study. Comparative activity profiling from different tissues showed seedlings to have elevated activity than other tissues. For kinetic characterization, the enzyme was partially purified by 12.3 fold using DEAE-Sephadex column and showed a narrow pH optimum of 7.0. In presence of saturated substrate concentration, the enzyme exhibited hyperbolic kinetics for both ADP and PEP with Km (Michaelis constant value) for PEP and ADP was found to be 178.5 and 96.45 μM respectively. ATP and citrate are the most significant allosteric effectors of the partially purified PK. Study on isozymes of PK resulted in a single band.

Effects of Matrix Filtration of Low‐Quality Boar Semen Doses on Sperm Quality

The aim of this work was to develop a method to enhance the sperm parameters of ejaculates with low sperm quality from Piétrain boars. Seminal doses were filtered through columns of DEAE Sephadex (length 2.5 +/- 0.5 cm), CM Sephadex (length 5 +/- 0.5 cm), glass wool (length 2 +/- 0.5 cm) or glass bead (length 10 +/- 0.5 cm), with an exit flow rate of 1 ml/40 s in all cases. For each male, 10 ml of the sperm cell-rich fraction diluted at 1 : 6 were filtered. Sperm quality was assessed before and after filtration. Sperm morphology, sperm motility and sperm concentration were determined using the computer program sca((R)) 2002 Production, and sperm viability was evaluated by fluorescence multistaining. Osmotic resistance test and hyperosmotic resistance test were used to determine the osmotic resistance of spermatozoa, whereas l-lactate production estimated the metabolic activity. Results showed a decrease of sperm concentration and osmotic resistance of spermatozoa after filtration in the four matrixes. However, an increase in the frequency of viable spermatozoa with intact acrosome after filtration in glass bead columns and an increase of morphologically normal spermatozoa after filtration in Sephadex CM-50, glass wool and glass bead columns were observed. Despite the decrease in the frequency of progressive motile spermatozoa, l-lactate production and mitochondrial sheath integrity maintained constant after filtration. Our findings indicate that column filtration is an effective method to enhance the sperm quality by selecting viable and morphologically normal spermatozoa without altering DNA, plasma membrane, mitochondrial sheath integrity or inducing premature acrosome reaction.

Effect of Actinidin on the Protein Solubility, Water Holding Capacity, Texture, Electrophoretic Pattern of Beef, and on the Quality Attributes of a Sausage Product

The objective of this study was to study the effect of actinidin, a sulfhydryl protease from kiwi fruit, on the protein solubility (nitrogen solubility index [NSI]), water holding capacity (WHC), texture, and SDS-PAGE pattern of beef and to evaluate the effect of pretreatment of beef with actinidin on the quality attributes of a sausage product. Actinidin was partially purified by precipitation with ammonium sulfate, followed by DEAE-Sephadex column chromatography. Actinidin significantly (P < 0.05) increased NSI and WHC of beef; the highest NSI and WHC (approximately 20% and 8% increase, respectively) was observed when beef was incubated with 0.9 unit enzyme/g beef. Texture analysis indicated increased tenderization (10% decrease in shear force) when slices of cattle beef were treated with actinidin at 37 degrees C for 2 h. SDS-PAGE results indicated appearance of several low molecular weight bands (<10 kDa) after treating beef with different levels of actinidin for 30 or 60 min. Slight changes in protein band in the range of 100 to 120 kDa and 13 to 25 kDa were also observed. Use of actinidin-tenderized beef significantly improved emulsion stability, texture, and organoleptic properties of the sausage product.

Demonstration That CobG, the Monooxygenase Associated with the Ring Contraction Process of the Aerobic Cobalamin (Vitamin B12) Biosynthetic Pathway, Contains an Fe-S Center and a Mononuclear Non-heme Iron Center

The ring contraction process that occurs during cobalamin (vitamin B(12)) biosynthesis is mediated via the action of two enzymes, CobG and CobJ. The first of these generates a tertiary alcohol at the C-20 position of precorrin-3A by functioning as a monooxygenase, a reaction that also forms a gamma lactone with the acetic acid side chain on ring A. The product, precorrin-3B, is then acted upon by CobJ, which methylates at the C-17 position and promotes ring contraction of the macrocycle by catalyzing a masked pinacol rearrangement. Here, we report the characterization of CobG enzymes from Pseudomonas denitrificans and Brucella melitensis. We show that both contain a [4Fe-4S] center as well as a mononuclear non-heme iron. Although both enzymes are active in vivo, the P. denitrificans enzyme was found to be inactive in vitro. Further analysis of this enzyme revealed that the mononuclear non-heme iron was not reducible, and it was concluded that it is rapidly inactivated once it is released from the bacterial cell. In contrast, the B. melitensis enzyme was found to be fully active in vitro and the mononuclear non-heme iron was reducible by dithionite. The reduced mononuclear non-heme was able to react with the oxygen analogue NO, but only in the presence of the substrate precorrin-3A. The cysteine residues responsible for binding the Fe-S center were identified by site-directed mutagenesis. A mechanism for CobG is presented.

HDAC1 Cooperates with C/EBPα in the Inhibition of Liver Proliferation in Old Mice

Epigenetic control of liver proliferation involves cooperation between transcription factors and chromatin-remodeling proteins. In this work, we found that the levels of HDAC1 (histone deacetylase 1) are increased in quiescent livers of old mice. The elevation of HDAC1 in liver is mediated by the RNA-binding protein CUGBP1. We found that the age-associated CUGBP1-eIF2 complex binds to the 5' region of HDAC1 mRNA and increases translation of HDAC1 in the liver. Further analyses showed that CUGBP1 also increases expression of HDAC1 in cultured cells, in the livers of CUGBP1 transgenic mice, and in the livers of mice injected with cyclin D3, which enhances the formation of the CUGBP1-eIF2 complex. In livers of old mice, HDAC1 interacts with the transcription factor C/EBPalpha and is recruited by this protein to E2F-dependent promoters as a component of high M(r) C/EBPalpha-Brm complexes. The recruitment of HDAC1 to c-Myc and FoxM1B promoters leads to deacetylation of histone H3 at Lys-9 on these E2F-dependent promoters. We show that HDAC1 is an important mediator of growth-inhibitory activity of C/EBPalpha and that small interfering RNA-mediated inhibition of HDAC1 reduces the ability of C/EBPalpha to inhibit cell proliferation. In addition, we have found that both elevation of HDAC1 and interaction of C/EBPalpha with HDAC1 are controlled by cyclin D3-dependent mechanisms. Treatment of old mice with growth hormone, which reduces cyclin D3 levels, leads to the reduction of the CUGBP1-eIF2 complex, normalization of HDAC1 levels, and inhibition of interactions of HDAC1 with C/EBPalpha-Brm complexes. Thus, our data demonstrate that translational elevation of HDAC1 in livers of old mice is involved in the assembly of high M(r) protein-protein complexes that inhibit liver proliferation.

Identification of gangliosides recognized by IgG anti-GalNAc-GD1a antibodies in bovine spinal motor neurons and motor nerves

The presence of immunoglobulin G (IgG)-type antibodies to the ganglioside, N-acetylgalactosaminyl GD1a (GalNAc-GD1a), is closely associated with the pure motor type of Guillain–Barré syndrome (GBS). In the present study, we isolated disialogangliosides from the motor neurons and motor nerves of bovine spinal cords by DEAE-Sephadex column chromatography. The disialoganglioside fraction contained GD1a, GD2, GD1b, and three gangliosides, designated X1, X2 and X3. Serum from a patient with axonal GBS with IgG anti-GalNAc-GD1a antibody yielded positive immunostaining with X1, X2, and X3. When isolated by preparative thin-layer chromatography (TLC), X1 migrated at the same position as GalNAc-GD1a from Tay-Sachs brain, suggesting that X1 is GalNAc-GD1a containing N-acetylneuraminic acid (NeuAc). TLC of isolated X2 revealed that it migrated between GD1a and GD2. On the other hand, X3 had a migratory rate on TLC between and GD1b and GT1b. Since both X2 and X3 were recognized by IgG anti-GalNAc-GD1a antibody, the results suggest that X2 is a GalNAc-GD1a species containing a mixture containing a NeuAc-and an N-glycolylneuraminic acid (NeuGc) species, and X3 is a GalNAc-GD1a species with two NeuGc. This evidence indicating the specific localization of GalNAc-GD1a and its isomers in spinal motor neurons should be useful in elucidating the pathogenic role of IgG anti-GalNAc-GD1a antibody in pure motor-type GBS.