DE-52 and DE-53 cellulose microcarriers. I. Growth of primary and established anchorage-dependent cells

DEAE cellulose anion exchangers having small ion exchange capacity (0.5 to 2.0 meq/g dry material) were tested for cell attachment kinetics and capacity to support growth anchorage-dependent cells. It was found that cells from established cell lines (BHK and MDCK) can grow to confluency on DEAE cellulose particles having exchange capacity of 1 and 2 meq/g dry materials, DE-52 and DE-53, respectively. On the other hand, chick embryo fibroblasts (primary cells) can grow only on DE-53 particles.     

Role of substrata in determining the growth topology of transformed and nontransformed cells in culture

Cylindrical DEAE cellulose anion exchangers (DE-53), generally used for chromatography, were found suitable as a substratum for cultivating cells. Embryonic avian and mammalian cells cultured on DE-53 microcarriers (MC) grow in multilayers, while the same embryonic cells when transformed by avian sarcoma virus (ASV) grow in monolayers. These patterns of cell growth differ from those of normal and transformed cells grown on conventional glass or plastic Petri dishes, or on beaded MC.Cells derived from established cell lines such as BHK, HeLa, L-929, MDCK, and VERO grow in monolayer on these MC. A human adenocarcinoma cell line is the only exception growing in a multilayer form. These results indicate that the ability of cultured cells to grow in multilayers, is determined not only by their state of transformation but also by the properties of the support on which they are cultured.     

Purification of Eimeria Sporozoites by DE-52 Anion Exchange Chromatography

An anion exchange column of DE-52 has been used to purify Eimeria sporozoites from a post-excystation mixture of oocysts, oocyst shells, sporocysts, sporocyst shells, and sporozoites. The mean recovery from several experiments was 94% and virtually all non-sporozoite material was removed. Infectivity studies in vitro with sporozoites showed that they were viable after purification and were at least as infectious as the unpurified sporozoites; furthermore, oocysts in the crude preparation could be recovered from the DE-52 cellulose by resuspending them in a 20% (w/v) sodium chloride solution.     

Effects of ion exchange capacities on attachment and growth of anchorage-dependent HeLa cell

An effect of ion exchange capacity of petri dish on cell attachment and growth of an anchorage-dependent animal cell was studied. We proposed a new idea to analyze cell attachment kinetics. As the experimental result of cell attachment to a petri dish, the attachment rate constant and the maximum number of attached cells both increase with increasing the ion exchange capacity. However, no direct correlation between the ion exchange capacity and the cell growth was observed. An influence of ion exchange capacity on the cell growth of initially attached cells onto the petri dish with different ion exchange capacities was examined. The specific growth rate of initially attached cells decreased with increasing the ion exchange capacity. The detrimental effect of the ion exchange capacity on the specific growth rate of the initially attached cells exists during the cell cultivation period. In the ion exchange capacity of 27 meq/m², the specific growth rate drastically decreased. It is considered that there is optimum value of the ion exchange capacity for the cell growth and attachment. It is concluded that the optimum ion exchange capacity is 18 meq/m².     

Detection of homozygotes and heterozygote carriers of GM2-gangliosidosis

11 patients with Tay-Sachs disease (TSD) and 4 patients with Sandhoff disease were identified using the methods of heat inactivation of hexosaminidase at 50 degrees C (3 and 4 hours) and electrofocusing on PAG-plates in the pH range 3.5-9.5. Ion exchange chromatography on DEAE cellulose DE-52 proved to be reliable for identification of heterozygotes in cases when the proband was not available. The incidence of TSD gene was estimated in 2 population samples--from the cities of Gomel and Kostroma. It was about 0.004 in the Gomel sample. No heterozygotes were detected in Kostroma.     

Influence of surface characteristics of cellulose carriers on ethanol production by immobilized yeast cells

Ethanol production was carried out by growing yeast cells immobilized on porous cellulose carriers. The effects of the chemical modification of cellulose carriers on cell immobilization and ethanol production were examined with respect to ion-exchange capacity and chemical structure. The ion-exchange capacity of 0·1 meq/g-carriers had no effect on immobilization but affected ethanol production by repeated batch cultures using immobilized yeast cells. Diethylaminoethyl was a suitable function group for immobilization and ethanol production. Ethanol productivity of the 10th batch cycle with diethylaminoethyl cellulose carriers was 23% greater than that of the first batch cycle.     

Biosorption of phosphate from synthetic wastewater by biosolids

The aim of this study was to determine the potential of phosphate (P) removal from wastewater by biosolids prepared by the immobilization of P-accumulating bacteria onto organic bentonite. Organic bentonite was prepared by treating bentonite clay with quaternary ammonium salt — cetyltrimetylammonium (CTA) bromide. Cation exchange capacity (CEC) of the bentonite was found to be 179.0 meq/100 g of the dry bentonite. The CTA occupied ca. 175% of the CEC. Modification of bentonite with CTA in amounts higher than 55% of the CEC resulted in the change of zeta potential of particles from negative to positive. Only in reactors containing organic bentonite samples occupied with 3.5 and 28% of the CEC was P efficiently removed from wastewater by combined adsorption and bacterial uptake in the biomass. Organic bentonite samples with higher CTA loadings (from 55 to 175% of the CEC) showed bactericidal effects. To enhance P removal from wastewater in the aerated biological system, biosolids consisting of P-accumulating bacteria and organic bentonite can be used, but special attention should be given to the configuration of sorbed CTA molecules and its potential desorption.     

Effects of the Commercial Flame Retardant Mixture DE-71 on Cytokine Production by Human Immune Cells

IntroductionAlthough production of polybrominated diphenyl ethers (PBDEs) is now banned, release from existing products will continue for many years. The PBDEs are assumed to be neurotoxic and toxic to endocrine organs at low concentrations. Their effect on the immune system has not been investigated thoroughly. We aimed to investigate the influence of DE-71 on cytokine production by peripheral blood mononuclear cells (PBMCs) stimulated with Escherichia Coli lipopolysaccharide (LPS) or phytohaemagglutinin-L (PHA-L).ResultsAt non-cytotoxic concentrations (0.01–10 μg/mL), DE-71 significantly enhanced secretion of IL-1β, IL-6, CXCL8, IL-10, and TNF-α (p<0.001–0.019; n = 6) from LPS-stimulated PBMCs. IFN-γ, TNF-α, IL-17A, and IL-17F (p = <0.001–0.043; n = 6) secretion were enhanced from PHA-L-stimulated PBMCs as well. Secretion of IL-1β, IL-2, IL-10, IL-8 and IL-6 was not significantly affected by DE-71.ConclusionsWe demonstrate an enhancing effect of DE-71 on cytokine production by normal human PBMCs stimulated with LPS or PHA-L ex vivo.     

Purification, some properties and the complete primary structures of two protease inhibitors (DE-3 and DE-4) from Macrotyloma axillare seed.

The Macrotyloma axillare plant, belonging to the Leguminosae family, is a perennial climbing or trailing herb 0.2--3.5 m long. The plant is indigenous to South Africa and it occurs in the warm dry northern parts of the Transvaal. It has been introduced into Australia, where the seed are used as animal food. Two protease inhibitors, DE-3 and DE-4, were purified from Macrotyloma axillare seed by gel filtration on Sephadex G-50 followed by ion-exchange chromatography on DEAE-cellulose. They each comprise 76 amino acid residues including 14 half-cystine residues. The complete primary structures of the two protease innibitors have been elucidated and their sequences are 67% identical. The inhibitor specificities, the sequences, the invariant amino acid residues and the reactive inhibitor sites of protease inhibitors DE-3 and DE-4 resemble the corresponding properties of the Bowman-Birk double-headed protease inhibitor group. The cysteine residues are in similar locations to those in protease inhibitors of known structure so they are presumed to link similarly.     

Purification and some properties of two proteinase inhibitors (DE-1 and DE-3) from Erythrina latissima (broad-leaved erythrina) seed

Two proteinase inhibitors, DE-1 and DE-3, were purified from Erythrina latissima seeds. Whereas DE-1 inhibits bovine chymotrypsin and not bovine trypsin, DE-3 inhibits trypsin but not chymotrypsin. The molecular weights and the amino acid compositions of the two inhibitors resemble the corresponding properties of the Kunitz-type proteinase inhibitors. The N-terminal primary structure of DE-3 showed homology with soybean trypsin inhibitor (Kunitz) and also with the proteinase inhibitors (A-II and B-II) from Albizzia julibrissin seed.     

Functional redundancy of the DE-1 and alpha A-CRYBP1 regulatory sites of the mouse alpha A-crystallin promoter.

Previous studies have implicated the DE-1 (-111/-106) and alpha A-CRYBP1 (-66/-57) sites for activity of the mouse alpha A-crystallin promoter in transiently transfected lens cells. Here we have used the bacterial chloramphenicol acetyltransferase (CAT) reporter gene to test the functional importance of the putative DE-1 and alpha A-CRYBP1 regulatory elements by site-specific and deletion mutagenesis in stably transformed alpha TN4-1 lens cells and in transgenic mice. FVB/N and C57BL/6 x SJL F2 hybrid transgenic mice were assayed for CAT activity in the lens, heart, lung, kidney, spleen, liver, cerebrum, and muscle. F0, F1, and F2 mice from multiple lines carrying single mutations of the DE-1 or alpha A-CRYBP1 sites showed high levels of CAT activity in the lens, but not in any of the non-lens tissues. By contrast, despite activity of the wild-type promoter, none of the mutant promoter/CAT constructs were active in the transiently transfected and stably transformed lens cells. The mice carrying transgenes with either site-specific mutations in both the DE-1 and alpha A-CRYBP1 sites or a deletion of the entire DE-1 and part of the alpha A-CRYBP1 site (-60/+46) fused to the CAT gene did not exhibit CAT activity above background in any of the tissues examined, including the lens. Our results thus indicate that the DE-1 and alpha A-CRYBP1 sites are functionally redundant in transgenic mice. Moreover, the present data coupled with previous transfection and transgenic mouse experiments suggest that this functional redundancy is confined to lens expression within the mouse and is not evident in transiently transfected and stably transformed lens cells, making the cultured lens cells sensitive indicators of functional elements of crystallin genes.     

Mineral nutrition, of cultured chlorophyllous cells of tobacco VII. Effects of the NH4/NO3 ratio and of Cl in the media on the growth and ionic balance of cells

NG, a strain of cultured tobacco cells of Nicotiana glutinosahad high growth rates and carboxylate contents (C—A) of100 to 130 meq/100 g of dry cells on media containing 42 meqNO₃/liter as the sole N source. (C—A) is the amount ofinorganic cations minus inorganic anions in meq per 100 g ofdry cells. NG, cultured on media containing NH₄ 10+NO₃ 42 in meq/liter,had lower growth rates and lower (C—A) values as comparedwith NG on media containing NO₃ as the sole N source. NG, cultured on media containing NH₄ 30+NO₃ 42 in meq/liter,had high growth rates and (A—C) values of 22 to 53 meq/100gof dry cells. In this case, the (A—C) content may correspondto organic cations, basic organic N compounds such as free asprotein-bound basic amino acids. The easily absorbed Cl mayhave been required maintain good growth conditions such as ionicbalance and a favorable pH in the cells. Thus cultured cells of Nicotiana glutinosa may have physiologicaladaptability against variations in a relatively wide range of|C—A| contents [|C—A| being the absolute valuesof (C—A)]. (Received May 15, 1980; )     

Preparation of highly purified C-phycoerythrin from marine cyanobacterium Pseudanabaena sp

C-phycoerythrin was isolated and purified from marine Pseudanabaena sp. using two step chromatographic methods. Phycobiliproteins in the marine Pseudanabaena was extracted in 100 mM phosphate buffer (pH 7.2) and precipitated by salting out. The precipitated C-phycoerythrin was purified by gel filtration with Sephadex G-150, and then it was purified by ion exchange chromatography on DEAE cellulose, which was developed by linear ionic strength gradients. Purified phycoerythrin showed absorption maxima at 568 and 541 nm, and displayed a fluorescence maximum at 578 nm. The absorbance ratio A???/A???, a criterion for purity (purity ratio) achieved was 6.86. It showed a single band on examination by polyacrylamide gel electrophoresis (PAGE). The polypeptide analysis of the purified C-phycoerythrin by SDS-PAGE demonstrated that it contained two chromophore-carrying subunits. The yield of purified C-phycoerythrin obtained was 13.6 mg/g of the cell dry weight with 47% of yield. Obtaining highly pure C-phycoerythrin allows one to evaluate its fluorescence properties for future applications in biochemical and biomedical research.     

Theileria parva: isolation of macroschizonts from in vitro propagated lymphoblastoid cells of cattle

A method for purifying macroschizonts of Theileria parva from bovine lymphoblastoid cells, propagated in vitro, was developed. This method involved three steps. First, the macroschizonts were liberated by disrupting host cells suspended in growth medium at 4 × 10⁶ cells/ml at 300–400 psi, using the Stansted cell disrupter. This yielded 80–90% disrupted cells while causing minimum damage to the macroschizonts. Second, the host cell nuclei were separated by (a) centrifuging the lysate at 300g for 60 min, (b) resuspending the pellet in 0.02 times the volume of initial host cell suspension in Leibovitz's L15 growth medium, and (c) lysing the host cell nuclei by adding nucleus-lysing buffer (NLB, containing 0.14 M Tris, 0.1 M HCl, 0.12 M glucose, and 0.5 M NaCl adjusted with NaOH to pH 7) to 0.2 times the volume of initial host cell suspension. The resulting chromatin precipitate was removed by adding DE-52 cellulose equilibrated with NLB and allowing the precipitate to sediment. Lastly, the final suspension obtained in the second step was applied on a DE-52 cellulose column which was equilibrated with the elution buffer (NLB with 10% fetal, or newborn, bovine serum, pH 7). Macroschizonts free of intact host cells and naked host cell nuclei were collected in the eluate. The protein yield was 2.7 mg per 10⁹ starting undisrupted host cells, which was 1.7% of the total starting protein.     

Endotoxin removal by anion-exchange polymeric matrix.

The effects of anion-exchange polymeric matrices on endotoxin removal from albumin and gamma-globulin solutions are evaluated. The positively charged cellulose acrylic media carrying DEAE or QAE functional groups remove significant amounts of endotoxin from tap water, but are less effective in protein solutions. With properly controlled pH levels and salt concentrations, the endotoxin level in a protein solution can be reduced; however, low endotoxin concentrations, less than 100 pg/ml, are more difficult to remove. The endotoxin removal capacity depends on the number of functional groups existing in the matrix, expressed as the number of milliequivalents (meq), and on the pH operable range, which is directly related to the pK alpha value of the matrix. The effects of pH and salt on endotoxin removal from albumin and gamma-globulin solutions by an anion-exchange polymeric matrix were evaluated statically in test tubes. In addition, a dynamic flow was performed under statically defined conditions on a 250-ml DEAE cartridge for the removal of endotoxin from albumin at a flow rate of 40 ml/min. A greater than 75% reduction in the endotoxin can be achieved, with protein loss occurring only in the early stage of removal. Such processes are useful for the reduction of endotoxin from biological solutions produced by natural sources or recombinant DNA technology.     

Improved Properties of Bacillus coagulans β‐Galactosidase through Immobilization

Thermostable β‐galactosidase from Bacillus coagulans RCS3 was purified by successive column chromatography using DEAE‐cellulose and Sephadex G‐50. Immobilization of the purified enzyme was studied with DEAE‐cellulose and calcium alginate. The efficiency of β‐galactosidase retention was 87 % with DEAE‐cellulose (17 mg protein/mL of matrix) and 80 % with calcium alginate (2.2 mg protein/g bead). Comparative studies of immobilization displayed a shift in the optimum temperature from 65 °C to 70 °C provoked by DEAE‐cellulose, although no effect was observed with calcium alginate. The heat inactivation curve revealed an improvement in the stability (t_1/2 of 14.5 h for the immobilized enzyme as compared to 2 h for the free enzyme at 65 °C) in a calcium alginate system. This immobilized enzyme has a wide pH stability range (6.5–11). β‐Galactosidase immobilized by DEAE‐cellulose and calcium alginate allowed a 57 and 70 % lactose hydrolysis, respectively, to be achieved within 48 h after repeated use for twenty times.     

细脚拟青霉菌丝体多糖纯化及组成分析

通过DE-52纤维素柱和Sephedex G-100葡聚糖凝胶柱,对细脚拟青霉Paecilomyces tenuipes菌丝体粗多糖进行纯化,得到PtPs1和PtPs2,应用离子色谱HPAEC-PAD对纯多糖PtPs1和PtPs2的单糖组成进行分析,证实均由葡萄糖、半乳糖和甘露糖三种单糖组成,经红外扫描确定PtPs1和PtPs2均为α型吡喃糖。     

Role of Cations in Accumulation and Release of Phosphate by Acinetobacter Strain 210A

Cells of the strictly aerobic Acinetobacter strain 210A, containing aerobically large amounts of polyphosphate (100 mg of phosphorus per g [dry weight] of biomass), released in the absence of oxygen 1.49 mmol of P_i, 0.77 meq of Mg²⁺, 0.48 meq of K⁺, 0.02 meq of Ca²⁺, and 0.14 meq of NH₄⁺ per g (dry weight) of biomass. The drop in pH during this anaerobic phase was caused by the release of 1.8 protons per PO₄³⁻ molecule. Cells of Acinetobacter strain 132, which do not accumulate polyphosphate aerobically, released only 0.33 mmol of P_i and 0.13 meq of Mg²⁺ per g (dry weight) of biomass but released K⁺ in amounts comparable to those released by strain 210A. Stationary-phase cultures of Acinetobacter strain 210A, in which polyphosphate could not be detected by Neisser staining, aerobically took up phosphate simultaneously with Mg²⁺, the most important counterion in polyphosphate. In the absence of dissolved phosphate in the medium, no Mg²⁺ was taken up. Cells containing polyphosphate granules were able to grow in a Mg-free medium, whereas cells without these granules were not. Mg²⁺ was not essential as a counterion because it could be replaced by Ca²⁺. The presence of small amounts of K⁺ was essential for polyphosphate formation in cells of strain 210A. During continuous cultivation under K⁺ limitation, cells of Acinetobacter strain 210A contained only 14 mg of phosphorus per g (dry weight) of biomass, whereas this element was accumulated in amounts of 59 mg/g under substrate limitation and 41 mg/g under Mg²⁺ limitation. For phosphate uptake in activated sludge, the presence of K⁺ seemed to be crucial.     

Immobilization of Acetobacter acoti on cellulose ion exchangers: adsorption isotherms

The adsorptive behavior of cells of Acetobacter aceti, ATCC 23746, on DEAE-, ECTEOLA-, TEAE-, and DEHPAE-cellulose ion exchangers in a modified Hoyer's medium at 30 degrees C was investigated. The maximum observed adsorption capacities varied from 46 to 64 mg dry wt/g resin. The Langmuir isotherm form was used to fit the data, since the cells formed a monolayer on the resin and exhibited saturation. The equilibrium constant in the Langmuir expression was qualitatively correlated with the surface charge density of the resin. The adsorption was also "normalized" by considering the ionic capacities of the resins. The exceptionally high normalized adsorption capacity of ECTEOLA-cellulose, 261 mg dry wt/meq, may be explained by an interaction between the cell wall and the polyglyceryl chains of the exchanging groups in addition to the electrostatic effects. The effect of pH on the bacterial adsorption capacity of ECTEOLA-, TEAE-, and phosphate-cellulose resins was studied and the pl of the bacteria was estimated to be 3.0.