L.D. Buckberry

A new Chinese hamster ovary cell line expressing α2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins

Chinese hamster ovary (CHO) cells are widely employed to produce glycosylated recombinant proteins. Our group as well as others have demonstrated that the sialylation defect of CHO cells can be corrected by transfecting the alpha2,6-sialyltransferase (alpha2,6-ST) cDNA. Glycoproteins produced by such CHO cells display both alpha2,6- and alpha2,3-linked terminal sialic acid residues, similar to human glycoproteins. Here, we have established a CHO cell line stably expressing alpha2,6-ST, providing a universal host for further transfections of human genes. Several relevant parameters of the universal host cell line were studied, demonstrating that the alpha2,6-ST transgene was stably integrated into the CHO cell genome, that transgene expression was stable in the absence of selective pressure, that the recombinant sialyltransferase was correctly localized in the Golgi and, finally, that the bioreactor growth parameters of the universal host were comparable to those of the parental cell line. A second step consisted in the stable transfection into the universal host of cDNAs for human glycoproteins of therapeutic interest, i.e. interferon-gamma and the tissue inhibitor of metalloproteinases-1. Interferon-gamma purified from the universal host carried 40.4% alpha2,6- and 59.6% alpha2,3-sialic acid residues and showed improved pharmacokinetics in clearance studies when compared to interferon-gamma produced by normal CHO cells.

The culture of neurons on silicon

We have investigated the adherence and subsequent viability of rat neuronal (B50) cells cultured directly on the nanostructured semiconductors porous silicon (PS), PECVD polycrystalline silicon and bulk silicon wafers. In contrast to our previous work on culturing CHO cells on nanostructured silicon, where the cell count was greatest on nanocrystalline PECVD Si (nc-Si) substrates, with B50 cells the optimum surface was PS, with much reduced cell viability observed for cells cultured on bulk or nc-Si. These preliminary studies indicate that PS and nc-Si offer advantages over bulk Si surfaces for neuron cell adherence and viability since they do not require coating with substances such as polylysine to support cell growth, PS is light-addressable, and nanostructured coatings can be applied to most object shapes, allowing flexibility in their deployment.

Nature of the Silicon-Animal Cell Interface

The paper reports the results of the study of cell culture growth at the surface of porous silicon. They show that porous and poly(nano)crystalline Si offer significant advantages over bulk Si surfaces for cell adherence and viability: these materials do not require coating with substances such as polylysine to support cell growth; porous Si is light-addressable because of photoluminescence and photovoltaic effects noted [Unal and Bayliss, J. Appl. Phys. 80, 3532 (1996)], allowing the potential for optical data transfer and less susceptibility to interference from external electronic equipment; finally nanostructured coatings can be applied to most object shapes, giving flexibility in their application.

Phosphate and cell growth on nanostructured semiconductors

Abstracts are not published in this journal

Na-butyrate increases the production and α2,6-sialylation of recombinant interferon-γ expressed by α2,6- sialyltransferase engineered CHO cells

A non-human like glycosylation pattern in human recombinant glycoproteins expressed by animal cells may compromise their use as therapeutic drugs. In order to correct the CHO glycosylation machinery, a CHO cell line producing recombinant human interferon- γ (IFN) was transformed to replace the endogenous pseudogene with a functional copy of the enzyme α2,6-sialyltransferase (α2,6-ST). Both the parental and the modified CHO cell line were propagated in serum-free batch culture with or without 1 mM sodium butyrate. Although Na-butyrate inhibited cell growth, IFN concentration was increased twofold. The IFN sialylation status was determined using linkage specific sialidases and HPLC. Under non- induced conditions, IFN expressed by α2,6-engineered cells contained 68% of the total sialic acids in the α2,6- conformation and the overall molar ratio of sialic acids to IFN was 2.3. Sodium butyrate addition increased twofold the molar ratio of total sialic acids to IFN and 82% of total sialic acids on IFN were in the α2,6-conformation. In contrast, no effect of the sodium butyrate was noticed on the sialylation of the IFN secreted by the α2,6-ST deficient parental cell line. This study deals for the first time with the effect of Na-butyrate on CHO cells engineered to produce human like sialylation.

Nanostructured semiconductors: compatibility with biomaterials

Abstract We carried out a study of the biocompatibility of silicon and germanium substrates of various porosities, by exposing them to a range of biological species. The aim of this is to discover more about the mutual compatibility of optoelectronics and living tissue. This preliminary study suggests that porous semiconductors have great potential in biological applications, however the toxicological implications have yet to be ascertained.

Novel Sources of Mammalian C-S Lyase Activity

The C‐S lysis of L‐cysteine conjugates is one biotransformation pathway which is responsible for the generation of mutagenic and cytotoxic metabolic species. Thirteen cysteine S‐conjugates were synthesized in our laboratories and incubated with aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) enzymes from porcine heart tissue. The C‐S lyase (CSL) activity for each enzyme‐substrate combination was determined.

The C-S lysis of L-cysteine conjugates by aspartate and alanine aminotransferase enzymes

One biotransformation pathway which is responsible for the generation of mutagenic and cytotoxic metabolites is that of the C-S lysis (CSL) of L-cysteine conjugates. Thirteen cysteine S-conjugates, synthesised in our labora tories, were incubated with porcine heart aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT), and the C-S lyase activity for each enzyme-sub strate combination was determined. ASAT and ALAT were shown to exhibit CSL activity. It was also demonstrated that this activity was inhibited in the presence of the pyri doxal phosphate (PLP)-dependent enzyme inhibitor amino(oxyacetic acid) (AOAA) confirming the pyridoxal phosphate dependent mechanism by which C-S lysis is known to take place. Since the activities of these enzymes are used as biomarkers for the assessment of organ dam age, the potential interaction of L-cysteine conjugates with them may suppress their activity through direct inhibition.

Cysteine conjugate β-lyase activity in three species of parasitic helminth

Abstract Living organisms employ a variety of metabolic pathways when detoxifying xenobiotic compounds, including the formation of cysteine S- conjugates via glutathione conjugation. However, cysteine conjugate β-lyase (CCBL) catalysed β-cleavage, of certain cysteine conjugates, is known to cause cytotoxicity. This study represents the first investigation into the expression of CCBL and other associated enzymes in helminth species. A survey of the three major groups of parasitic helminths [cestodes ( Moniezia expansa ), digeneans ( Fasciola hepatica ) and nematodes ( Necator americanus , Heligmosomoides polygyrus )] has been made. The presence of CCBL enzymes within Moniezia expansa , Necator americanus and Heligmosomoides polygyrus has been established. Each species was screened for γ-glutamyl transpeptidase activity and transaminase activity towards l -aspartate, l -alanine, l -albizziin and l -phenylalanine. Aspartate and alanine aminotransferase activity were detected in all four species tested. γ-Glutamyl transpeptidase activity was only detected in Moniezia expansa and Necator americanus .

Purification and characterisation of a novel cysteine conjugate β-lyase from the tapeworm Moniezia expansa.

The paper presents the first report of the purification of an invertebrate cysteine conjugate beta-lyase (CCBL). CCBL activity was shown to predominate within the cytosolic fraction of tissue from the tapeworm Moniezia expansa. The monomeric cytosolic enzyme was isolated with a M(r) of 72 kDa and co-purified with transaminase activity towards L-aspartate. The substrate profile for M. expansa CCBL is different from that of mammalian CCBLs. Exploiting the differences in mammalian and parasite substrate profiles will facilitate the development of helminth targeted conjugates which will not be activated by host (mammalian) CCBLs.