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A novel monoclonal antibody was raised by immunization of mice with colorectal tumor cell lines. The fusion was screened by immunohistochemistry for binding to primary colorectal tumors. Subsequent analysis on primary disaggregated colorectal tumors show that the antibody recognizes a cell surface antigen expressed by the majority of colorectal tumors. Antigen characterization has shown that the antibody recognizes a sialyltetraosylceramide but does not bind to GM1, GD1a, GT1b, or sialyl LewisX antigens. Binding to a frozen panel of tumor and normal tissue sections revealed that the antigen was also strongly expressed on esophageal, gastric, and endometrial tumors. Its normal tissue distribution was largely restricted to moderate staining of large intestine. Surprisingly, SC104 antibody directly induces tumor cell death without the need for immune effector cells or complement. This may be related in part to its homophilic binding properties that allow cross-linking of antibody and receptors on the cell surface. Caspase activation can be detected following SC104 treatment of colorectal cells, and cotreatment with caspase inhibitors has been shown to inhibit cell death. This suggests that SC104 induces death by a classic apoptotic pathway. Furthermore, SC104 antibody shows additive killing with complement and 5-fluorouracil/leucovorin in vivo, suggesting a new therapeutic approach for this class of antibodies. (Cancer Res 2006; 66(11): 5901-9)

PS has been shown to be an excellent candidate biomaterial following studies showing its biostability and non-toxicity. These favourable properties, coupled with the ease of its topographical manipulation, and its optoelectronic properties, make it an ideal material for the design of biologically interfaced devices (BIDs). Possible potential applications for PS in BIDs falls into three main areas: (i) in vitro biosensors, (ii) the development of intelligent implantible medical devices and (iii) biologically interfaced neural networks. The majority of these applications rely on the ability of PS to directly interface with living cells. In order to achieve this the cells must adhere to the solid-state device in a pattern appropriate to the circuitry. Here we report the use of confocal microscopy to image B50 cells on PS substrates in order to obtain cell patterning information. We also report the quantification of silicic acid and silica toxicity on B50 cells in culture, a study confirming that suggested by-products of PS manufacture are not toxic. Finally, preliminary findings on the effect of nerve growth factor (NGF) on the morphology of B50 cells in culture are presented.

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 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.

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.

L-Cysteine conjugates are normally metabolised via N-acetylation to produce a mercapturic acid. However, a recently identified metabolic route (C-S lysis) may lead to the generation of an unstable thiol which has been demonstrated to be responsible for toxicity in various mammalian species.Human Chang liver cells were challenged with a number of established L-cysteine conjugates. The cellular toxicity of these compounds was determined using a range of assay procedures, which provided differing information, depending on the assay method used. These observations were then investigated in order to establish which system would provide the most reliable indication of C-S lyase toxicity and whether any information on the mechanism of action could be obtained by these assay methods.

It is now well recognized that cysteine conjugates are sometimes responsible for the mutagenic and cytotoxic consequences which occur following the ingestion of halocarbons. These toxic reactions arise from the generation of reactive metabolites via the cleavage of the C-S bond in cysteine conjugates. The enzymes which facilitate this reaction on cysteinyl conjugate metabolites are now known as C-S lyases. The products of the C-S lyase catalysis of cysteine conjugates are a reactive thiol, pyruvic acid and ammonia. Here we report the development of a sensitive fluorometric hplc assay for pyruvic acid using the derivatising agent 1,2-phenylenediamine. It was found that using this assay CSL activity could be detected in a porcine C-S lyase preparation. It was also observed that this assay was approximately ten-fold more sensitive than the existing spectrophotometric assay. It is now well recognized that cysteine conjugates are sometimes responsible for the mutagenic and cytotoxic consequences which occur following the ingestion of halocarbons. These toxic reactions arise from the generation of reactive metabolites via the cleavage of the C-S bond in cysteine conjugates. The enzymes which facilitate this reaction on cysteinyl conjugate metabolites are now known as C-S lyases. The products of the C-S lyase catalysis of cysteine conjugates are a reactive thiol, pyruvic acid and ammonia. Here we report the development of a sensitive fluorimetric hplc assay for pyruvic acid using the derivatizing agent 1,2-phenylenediamine. It was found that using this assay CSL activity could be detected in a porcine C-S lyase preparation. It was also observed that this assay was approximately ten-fold more sensitive than the existing spectrophotometric assay.

Kynurenine aminotransferase and glutamine transaminase K and L activities were assessed in order to identify a physiological role for human hepatic cytosolic CS lyase (CSL), as the CC lyase kynureninase, which co-purifies with rat hepatic CSL, has been shown not to be human hepatic CSL. Kynurenic acid production supports an aminotransferase role for this CSL. Kynurenine aminotransferase and glutamine transaminase K and L activities were assessed in order to identify a physiological role for human hepatic cytosolic C-S lyase (CSL), as the C-C lyase kynureninase, which copurifies with rat hepatic CSL, has shown not to be human hepatic CSL. Kynurenic acid production supports an aminotransferase role for this CSL.

Partially purified human hepatic cytosolic and mitochondrial fractions have been investigated for evidence of C-S lyase (CSL) activity. CSL activity has been characterized with synthetic aliphatic and aromatic L-cysteine conjugates. Preliminary structure-activity relationship studies have shown that aliphatic and aromatic L-cysteine conjugates are substrates.

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. © 1997 Elsevier Science S.A.

The paper presents the first report of the purification of an invertebrate cysteine conjugate β-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.

Porous silicon has now been used in a range of in vitro and in vivo bioapplications, from implant coatings to cell culture substrates and biosensors [1, 2, 3, 4, 5]. These widening applications are in part due to the large specific surface of porous silicon, the nano and micro-machinability of silicon, and the potential of nano and microstructured silicon to be used in novel devices due to the greatly modifiable optoelectronic properties. However there is also a growing body of literature which testifies the biocompatibility of porous silicon. Thus there appears to be many exciting biological areas in which porous silicon could play a major role. These include medical, environmental and computing technologies, and generally will require the immobilization of cells and enzymes in specific regions on porous silicon substrates. Once this has been achieved, the porous silicon can be built into, or supplied with, the relevant device architectures. However much background work needs to be done for these first steps to be realised. This itself requiring an understanding at the molecular level of interactions at the porous silicon surface. This interface between a cell and an inorganic material may seem straightforward at the micron level. It is in fact a dynamic system at the molecular level. Firstly, the substrate usually exhibits an oxide on the surface, due to exposure to air and the sterilization pre-treatment. Secondly, it is in a physiological environment, and generally proteins in the serum will coat the surface. In addition to this, it is necessary to ensure that the correct biosystem is adhered to the surface at the correct location. To go further in the deployment of porous silicon, therefore, it is necessary first to gain an understanding of the current techniques used in the research and development of biomaterials. And it is necessary to assess their applicability for the case of porous silicon. In particular the requirements for biomaterials need to be described, and the methods for controlling the interactions of biomolecules with biomaterials need to be clarified.

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 .

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. ASAT and ALAT were shown to exhibit CSL activity and it was also demonstrated that this activity was inhibited in the presence of the pyridoxal phosphate-dependent enzyme inhibitor amino(oxyacetic acid) confirming the pyridoxal phosphate-dependent mechanism by which C-S lysis is known to take place. This finding has potentially important implications for the risk assessment of compounds which produce L-cysteine conjugates during their biotransformation.

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.

C-S lyase enzymes catalyse the generation of mutagenic and/or cytotoxic thiols from cysteine conjugated xenobiotics. These cysteine conjugates are produced subsequent to glutathione conjugations as a metabolic step in the mercapturic acid pathway, traditionally thought of as a pathway solely associated with detoxification. Human Chang liver (HCL) cells were challenged with a range of cysteine conjugates demonstrated to be substrates for human hepatic C-S lyases. The cellular toxicity of these compounds was determined and it was observed that the rank order of substrate toxicity obtained for the HCL cells followed the rank order of C-S lyase activity of the substrates in a freshly isolated mitochondrial fraction of human tissue. The presence of C-S lyase activity was also established in this cell line.

The C-S lyase enzymes are responsible for the generation of mutagenic and cytotoxic metabolites via aberrant drug-metabolising pathways in mammalian tissues. We have examined human hepatic cytosolic, mitochondrial and microsomal fractions for evidence of C-S lyase activity. The cytosolic enzyme was purified using fast protein liquid chromatography over FFQ Sepharose, Mono P and Superose 12. An homogeneous protein (monitored by SDS-PAGE) was obtained following purification, and an 11-fold increase in C-S lyase specific activity was observed. The molecular weight of the enzyme was found to be 37 kDa in denaturing conditions, 82.3 kDa in non-denaturing conditions, and the C-S lyase activity was shown to co-purify with kynurenine aminotransferase activity when the transaminase activity of the enzyme was examined with kynurenine as the substrate.

The enzyme cysteine conjugate /.I-lyase (C-S lyase, EC 4.4.1.13) from rat renal tissue has been identified as a transaminase: glutamine transaminase K (GTK) [l]. This is one member of a family of enzymes which effect a transamination reaction on amino acid substrates. Other members of this family include glutamine transaminase L (GTL, isolated from rat liver, but which has a structure-activity profile broadly overlapping that of GTK), kynurenine aminotransferase (KAT), and aspartate aminotransferase. Rat hepatic cytosolic C-S lyase co-purifies as kynureninase, an enzyme which is involved in anthranilic acid biosynthesis [2], but antibodies raised against this purified protein did not crossreact with the renal cytosolic enzyme (i.e. with GTK). In one study with human hepatic cytosolic C-S lyase [3], kynurenine was not a substrate for the enzyme. These workers did not establish whether the C-S lyase enzyme had a physiological role, and if it co-purified with a protein which had been previously characterized. We have recently established that human hepatic cytosolic C-S lyase co-purifies as an aminotransferase: KAT [4]. In this article, we discuss the structureactivity relationships displayed by human C-S lyases obtained from both the cytosolic and mitochondrial sub-cellular fractions of kidney, liver and lung tissue. Elsewhere