Heterogeneous glycosylation of cationic peanut peroxidase

Cationic peanut peroxidase (CPrx) from a cell suspension culture is N-glycosylated at Asn60, Asn144, and Asn185. All three N-glycans are complex type and galactose rich, and show heterogeneity in length and ConA (concanavalin A) binding property. The glycan heterogeneity causes a polymorphism of the enzyme. Based on its behavior on ConA columns, CPrx can be grouped into two fractions: nonbinding (CPrx−) and binding (CPrx+) types. A synchronously cosecreted β-galactosidase has been discovered in the culture medium; there are two isozymes of 60 kDa (pI 7.3) and 66 kDa (pI 7.6). This β-galactosidase has been partially purified by a combination of ion-exchange and size-exclusion chromatographies and preparative isoelectrofocusing. In vitro experiments indicate that the cosecreted β-galactosidase is able to convert peroxidase from CPrx− to CPrx+ and may, to some extent, contribute to the glycan heterogeneity of peroxidase in the cell culture.Key words: peroxidase, peanut, glycan, heterogeneity, β-galactosidase.

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In Vitro Removal of Ochratoxin A by Wine Lactic Acid Bacteria

Journal of Food Protection ◽

10.4315/0362-028x-70.9.2155 ◽

2007 ◽

Vol 70 (9) ◽

pp. 2155-2160 ◽

Cited By ~ 47

Author(s):

VINCENZO DEL PRETE ◽

HECTOR RODRIGUEZ ◽

ALFONSO V. CARRASCOSA ◽

BLANCA de las RIVAS ◽

EMILIA GARCIA-MORUNO ◽

...

Keyword(s):

Lactic Acid ◽

Lactic Acid Bacteria ◽

Ochratoxin A ◽

High Performance ◽

Culture Medium ◽

Degradation Products ◽

Cell Binding ◽

A Cell ◽

In Vitro Interaction

A study was carried out to determine the in vitro interaction between ochratoxin A (OTA) and wine lactic acid bacteria (LAB). Fifteen strains belonging to five relevant oenological LAB species were grown in liquid synthetic culture medium containing OTA. The portion of OTA removed during the bacterial growth was 8 to 28%. The OTA removed from the supernatants was partially recovered (31 to 57%) from the bacterial pellet. Cell-free extracts of three representative strains were produced by disrupting cells in a French pressure cell. The ability of crude cell-free extracts to degrade OTA was studied. OTA was not degraded by cell-free extracts of wine LAB strains, and no degradation products of OTA were detected in the high-performance liquid chromatograms of the methanol extract of the bacterial pellet. On the basis of these results, we conclude that OTA removal by wine LAB is a cell-binding phenomenon. The chemistry and the molecular basis of OTA binding to wine LAB remains unknown.

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Comparison of Extracellular and Intracellular Potency of Botulinum Neurotoxins

Infection and Immunity ◽

10.1128/iai.00552-06 ◽

2006 ◽

Vol 74 (10) ◽

pp. 5617-5624 ◽

Cited By ~ 7

Author(s):

Fang Cai ◽

Carrie B. Adrion ◽

James E. Keller

Keyword(s):

Culture Medium ◽

Reaction Model ◽

Cultured Neurons ◽

Total Inhibition ◽

Botulinum Neurotoxins ◽

A Cell ◽

Proteolytic Activities ◽

Living Neurons ◽

Subsequent Addition

ABSTRACT Levels of botulinum neurotoxin (BoNT) proteolytic activity were compared using a cell-free assay and living neurons to measure extracellular and intracellular enzymatic activity. Within the cell-free reaction model, BoNT serotypes A and E (BoNT/A and BoNT/E, respectively) were reversibly inhibited by chelating Zn2+ with N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). BoNT/E required relatively long incubation with TPEN to achieve total inhibition, whereas BoNT/A was inhibited immediately upon mixing. When naïve Zn2+-containing BoNTs were applied to cultured neurons, the cellular action of each BoNT was rapidly inhibited by subsequent addition of TPEN, which is membrane permeable. Excess Zn2+ added to the culture medium several hours after poisoning fully restored intracellular toxin activity. Unlike TPEN, EDTA irreversibly inhibited both BoNT/A and -E within the cell-free in vitro reaction. Excess Zn2+ did not reactivate the EDTA-treated toxins. However, application of EDTA-treated BoNT/A or -E to cultured neurons demonstrated normal toxin action in terms of both blocking neurotransmission and SNAP-25 proteolysis. Different concentrations of EDTA produced toxin preparations with incrementally reduced in vitro proteolytic activities, which, when applied to living neurons showed undiminished cellular potency. This suggests that EDTA renders the BoNT proteolytic domain conformationally inactive when tested with the cell-free reaction, but this change is corrected during entry into neurons. The effect of EDTA is unrelated to Zn2+ because TPEN could be applied to living cells before or after poisoning to produce rapid and reversible inhibition of both BoNTs. Therefore, bound Zn2+ is not required for toxin entry into neurons, and removal of Zn2+ from cytosolic BoNTs does not irreversibly alter toxin structure or function. We conclude that EDTA directly alters both BoNTs in a manner that is independent of Zn2+.

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Efficient Delivery of dsRNA and DNA in Cultured Silkworm Cells for Gene Function Analysis Using PAMAM Dendrimers System

Insects ◽

10.3390/insects11010012 ◽

2019 ◽

Vol 11 (1) ◽

pp. 12

Author(s):

Chenchen Lu ◽

Zhiqing Li ◽

Li Chang ◽

Zhaoming Dong ◽

Pengchao Guo ◽

...

Keyword(s):

Gene Function ◽

Culture Medium ◽

Target Genes ◽

High Efficiency ◽

Function Analysis ◽

Binding Capacity ◽

Pamam Dendrimers ◽

Binding Property ◽

Gene Function Analysis

Polyamidoamine (PAMAM) dendrimers are emerging as intriguing nanovectors for nucleic acid delivery because of their unique well-defined architecture and high binding capacity, which have been broadly applied in DNA- and RNA-based therapeutics. The low-cost and high-efficiency of PAMAM dendrimers relative to traditional liposomal transfection reagents also promote their application in gene function analysis. In this study, we first investigated the potential use of a PAMAM system in the silkworm model insect. We determined the binding property of G5-PAMAM using dsRNA and DNA in vitro, and substantially achieved the delivery of dsRNA and DNA from culture medium to both silkworm BmN and BmE cells, thus leading to efficient knockdown and expression of target genes. Under treatments with different concentrations of G5-PAMAM, we evaluated its cellular cytotoxicity on silkworm cells, and the results show that G5-PAMAM had no obvious toxicity to cells. The presence of serum in the culture medium did not affect the delivery performance of DNA and dsRNA by G5-PAMAM, revealing its convenient use for various purposes. In conclusion, our data demonstrate that the PAMAM system provides a promising strategy for delivering dsRNA and DNA in cultured silkworm cells and promote its further application in individuals.

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Effects of stage of oestrous cycle and progesterone supplementation during culture on maturation of canine oocytes in vitro

Reproduction ◽

10.1530/rep.0.1260501 ◽

2003 ◽

pp. 501-508 ◽

Cited By ~ 40

Author(s):

LA Willingham-Rocky ◽

K Hinrichs ◽

ME Westhusin ◽

DC Kraemer

Keyword(s):

Selection Criteria ◽

In Vitro Maturation ◽

Culture Medium ◽

Oestrous Cycle ◽

In Vitro Experiments ◽

Meiotic Arrest ◽

Treatment Groups ◽

Key Factor ◽

Progesterone Supplementation

The aim of this study was to evaluate the effect of progesterone supplementation and stage of oestrous cycle on in vitro maturation (IVM) of canine oocytes. Oocytes were cultured in medium supplemented with 0, 2000, 4000 or 8000 ng progesterone ml(-1) (Expt 1; n=274 oocytes) or 0, 20, 200 or 2000 ng progesterone ml(-1) (Expt 2; n=789 oocytes). In Expt 3, oocytes (n=1202) were cultured in a bi-phasic system of meiotic arrest followed by IVM, both in the presence of 0, 20, 200 or 2000 ng progesterone ml(-1). Rates of meiotic resumption for Expt 1 ranged from 40.0% to 58.5%; there were no significant differences among groups. In Expt 2, rate of meiotic resumption was significantly lower in the 2000 ng progesterone ml(-1) treatment (35.5%) compared with the 200 ng progesterone ml(-1) treatment (54.0%; P<0.05). There were no significant differences in rates of maturation to metaphase II among treatments in Expt 1 (1.8-8.6%) or Expt 2 (8.4-14.7%); however, oocytes collected from ovaries of bitches in oestrus and dioestrus had higher rates of maturation to metaphase II than did oocytes from bitches at pro-oestrus or anoestrus (P<0.01). In Expt 3, no differences were observed in rates of maturation among treatment groups. Rates of maturation to metaphase II of oocytes from bitches in dioestrus were significantly higher than those from bitches in pro-oestrus (P<0.01). These results indicate that supplementation of culture medium with progesterone either during maturation or during meiotic arrest before maturation does not increase the rate of IVM of canine oocytes. However, stage of oestrous cycle is a key factor in the selection criteria for meiotically competent canine oocytes for use in in vitro experiments.

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Intein-mediated backbone cyclization of entolimod confers enhanced radioprotective activity in mouse models

10.7287/peerj.preprints.26803v1 ◽

2018 ◽

Author(s):

Bingyu Ye ◽

Wenlong Shen ◽

Minglei Shi ◽

Yan Zhang ◽

Cunshuan Xu ◽

...

Keyword(s):

Ion Exchange ◽

Clinical Investigation ◽

Affinity Purification ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Size Exclusion ◽

Radioprotective Activity ◽

Backbone Cyclization

Background. Entolimod is a Salmonella enterica flagellin derivate. Previous work has demonstrated that entolimod effectively protects mice and non-human primates from ionizing radiation. However, it caused a “flu-like” syndrome after radioprotective and anticancer clinical application, indicating some type of immunogenicity and toxicity. Cyclization is commonly used to improve the in vivo stability and activity of peptides and proteins. Methods. We designed and constructed cyclic entolimod using split Npu DnaE intein with almost 100% cyclization efficiency. We adopted different strategies to purify the linear and circular entolimod due to their different topologies. Results. After Ni-chelating affinity purification, the linear and circular entolimod were purified by size-exclusion and ion-exchange chromatography, respectively. Compared with linear entolimod, the circular entolimod showed significantly increased both the in vitro NF-κB signaling and in vivo radioprotective activity in mice. Discussions/Conclusions. Our data indicates that circular entolimod might be a good candidate for further clinical investigation.

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Effect of Nanostructured Scaffold on Human Adipose-Derived Stem Cells: Outcome of In Vitro Experiments

Nanomaterials ◽

10.3390/nano10091822 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1822 ◽

Cited By ~ 1

Author(s):

Marina Borgese ◽

Ludovica Barone ◽

Federica Rossi ◽

Mario Raspanti ◽

Roberto Papait ◽

...

Keyword(s):

Stem Cells ◽

Culture Medium ◽

Quantitative Polymerase Chain Reaction ◽

Enzyme Linked Immunosorbent Assay ◽

Adipose Derived Stem Cells ◽

In Vitro Experiments ◽

Fatty Acid Binding ◽

Beneficial Effects ◽

Angiogenesis Process

This work is addressed to provide, by in vitro experiments, results on the repercussion that a nanostructured scaffold could have on viability, differentiation and secretion of bioactive factors of human adipose-derived stem cells (hASCs) when used in association to promote angiogenesis, a crucial condition to favour tissue regeneration. To achieve this aim, we evaluated cell viability and morphology by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and microscopy analysis, respectively. We also investigated the expression of some of those genes involved in angiogenesis and differentiation processes utilizing quantitative polymerase chain reaction (qPCR), whereas the amounts of Vascular Endothelial Growth Factor A, Interleukin 6 and Fatty Acid-Binding Protein 4 secreted in the culture medium, were quantified by enzyme-linked immunosorbent assay (ELISA). Results suggested that, in the presence of the scaffold, cell proliferation and the exocytosis of factors involved in the angiogenesis process are reduced; by contrast, the expression of those genes involved in hASC differentiation appeared enhanced. To guarantee cell survival, the construct dimensions are, generally, smaller than clinically required. Furthermore, being the paracrine event the primary mechanism exerting the beneficial effects on injured tissues, the use of conditioned culture medium instead of cells may be convenient.

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Silicate and Calcium Ions Releasing Biomaterials for Bone Reconstruction

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.493-494.561 ◽

2011 ◽

Vol 493-494 ◽

pp. 561-565

Author(s):

Jin Nakamura ◽

Akiko Obata ◽

Julian R. Jones ◽

Toshihiro Kasuga

Keyword(s):

Calcium Ions ◽

Culture Medium ◽

Fetal Bovine Serum ◽

Poly Lactic Acid ◽

Cell Culture Medium ◽

A Cell ◽

Fetal Bovine ◽

Mouse Osteoblast ◽

Phosphate Deposits

Siloxane-containing vaterite (SiV) / poly (lactic acid) hybrid (SiPVH) beads with the releasability of silicate and calcium ions were prepared with an electrospraying method. According to the increase in the silicon content of the SiV, the amount of silicate ion released from the resulting beads also increased. When the beads were soaked in a cell culture medium, proteins derived from fetal bovine serum were adsorbed on their surfaces. Cell adhesion tests were also performed on the beads with using mouse osteoblast-like cell line (MC3T3-E1) in vitro. After 5 days of culturing, the cells adhered and spread well to cover the surface of the beads. In the localized area, agglomerated cells were observed to combine with cauliflower-shaped calcium phosphate deposits.

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108 IS THE BINDING OF COXIELLA BURNETII TO THE ZONA PELLUCIDAE FOLLOWING IN VITRO INFECTION OF IN VITRO-PRODUCED GOAT EMBRYOS CONCENTRATION DEPENDENT?

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab108 ◽

2017 ◽

Vol 29 (1) ◽

pp. 162

Author(s):

F. Fieni ◽

A. Alsaleh ◽

J. M. de Souza-Fabjan ◽

P. Mermillod ◽

E. Corbin ◽

...

Keyword(s):

Coxiella Burnetii ◽

Zona Pellucida ◽

Pathogen Transmission ◽

Binding Property ◽

In Vitro Experiments ◽

Conventional Pcr ◽

Bacterial Concentrations ◽

Spontaneous Infection

Previous experiments using in vitro infection have shown that at concentrations of 109 bacteria/mL, Coxiella burnetii strongly adheres to the zona pellucidae (ZP) of caprine embryos produced in vitro or in vivo (Alsaleh et al., 2013). However, spontaneous infection results in bacterial concentrations of between 106 and 107 bacteria/mL (Rodolakis, 2006; Alsaleh et al., 2011). The aim of this study was to determine whether the concentration of Coxiella burnetii affected its ability to bind to the ZP in vitro. A total of 120 ZP-intact 8- to 16-cell embryos, produced in vitro from ovaries collected at slaughter, were infected with Coxiella burnetti (strain CbC1) produced via ovoculture at 109 mL−1 (3 batches of 10 embryos), 107/mL (5 batches of 10 embryos), 105 mL−1 (3 batches of 8 embryos). After overnight incubation at 37°C in 5% CO2, the embryos were recovered and washed in batches, in 10 successive baths of PBS with 5% FCS, in accordance with International Embryo Technology Society guidelines. The 10 wash baths were collected separately and centrifuged for 1 h at 13,000 × g. The presence of C. burnetii was determined by conventional PCR in each batch of embryos and in the pellets of the 10 wash baths (Table 1). As demonstrated previously, Coxiella DNA was detected in embryo batches after 10 washes when a concentration of 109 bacteria/mL was used for in vitro infection, but this binding property did not occur at lower bacterial concentrations. We can conclude that the attachment of Coxiella burnetii to the zona pellucida of in vitro-produced embryos is concentration dependent. This finding illustrates the limitations of in vitro experiments to study the risk of pathogen transmission via embryo transfer. Table 1. Detection of Coxiella burnetii (CB) in successive embryo washing baths and batches of 8 to 10 infected ZP-intact 8- to 16-cell embryos after 10 wash cycles, using conventional PCR (C-PCR), as a function of the concentration of CB used for in vitro infection and determined by quantitative PCR (Q-PCR)

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Retrovirally transduced antisense sequences stably suppress P210BCR-ABL expression and inhibit the proliferation of BCR/ABL-containing cell lines

Blood ◽

10.1182/blood.v81.2.502.502 ◽

1993 ◽

Vol 81 (2) ◽

pp. 502-509 ◽

Cited By ~ 46

Author(s):

P Martiat ◽

P Lewalle ◽

AS Taj ◽

M Philippe ◽

Y Larondelle ◽

...

Keyword(s):

Culture Medium ◽

Philadelphia Chromosome ◽

K562 Cells ◽

Cellular Growth ◽

Growth Properties ◽

Bone Marrow Cultures ◽

A Cell ◽

Oncogenic Protein ◽

B10 Cells

Abstract There is now strong evidence that the BCR-ABL gene product of the Philadelphia chromosome (P210) plays a crucial role in the pathogenesis of chronic myeloid leukemia (CML). We have previously shown that introduction of antisense oligonucleotides into K562 cells could transiently block the expression of P210 and specifically inhibit cellular growth in culture. In this report, we describe the use of a retroviral vector to introduce selected antisense and sense sequences, first into murine B10 cells, previously rendered interleukin-3 (IL-3) independent by transfection of BCR-ABL sequences, and second into K562 cells. The antisense transcripts generated under the control of MoMLV promoter specifically killed B10 cells in the absence of IL-3 and inhibited P210 expression almost completely. In K562 cells, the antisense sequences led to a dramatic reduction of P210 expression and increased their doubling time by more than twofold. This effect was not reversed by the addition of exogenous IL-3 to the culture medium. Control HeLa or HL60 cells infected with the same constructs did not show any change in proliferation rate, despite abrogation of the normal BCR gene products. Rather unexpectedly, P210 suppression was not lethal in K562 cells, showing that such a cell line does not rely entirely on the expression of P210 for surviving, but depends on it as far as growth properties are concerned. We conclude that this approach can successfully achieve stable suppression of the oncogenic protein P210 and may be used to study further the mechanisms by which P210 is transforming cells. The effect on fresh CML cells in bone marrow cultures remains to be assessed before we can tell whether this technique may be used for selective suppression of leukemic hematopoiesis in vitro.

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