Ross L. Prestidge

Cloning and Biological Activity of Epigen, a Novel Member of the Epidermal Growth Factor Superfamily

High throughput sequencing of a mouse keratinocyte library was used to identify an expressed sequence tag with homology to the epidermal growth factor (EGF) family of growth factors. We have named the protein encoded by this expressed sequence tag Epigen, for epithelial mitogen. Epigen encodes a protein of 152 amino acids that contains features characteristic of the EGF superfamily. Two hydrophobic regions, corresponding to a putative signal sequence and transmembrane domain, flank a core of amino acids encompassing six cysteine residues and two putativeN-linked glycosylation sites. Epigen shows 24–37% identity to members of the EGF superfamily including EGF, transforming growth factor α, and Epiregulin. Northern blotting of several adult mouse tissues indicated that Epigen was present in testis, heart, and liver. Recombinant Epigen was synthesized in Escherichia coli and refolded, and its biological activity was compared with that of EGF and transforming growth factor α in several assays. In epithelial cells, Epigen stimulated the phosphorylation of c-erbB-1 and mitogen-activated protein kinases and also activated a reporter gene containing enhancer sequences present in the c-fos promoter. Epigen also stimulated the proliferation of HaCaT cells, and this proliferation was blocked by an antibody to the extracellular domain of the receptor tyrosine kinase c-erbB-1. Thus, Epigen is the newest member of the EGF superfamily and, with its ability to promote the growth of epithelial cells, may constitute a novel molecular target for wound-healing therapy.

The ability of heat-killed Mycobacterium vaccae to stimulate a cytotoxic T-cell response to an unrelated protein is associated with a 65 kilodalton heat-shock protein

Exogenous antigens are generally presented by Class II major histocompatibility (MHC) molecules. When administered with an adjuvant, however, they are capable of inducing a CD8+ T‐cell response where antigen recognition is associated with Class I MHC. Accordingly, immunization with soluble ovalbumin (OVA) alone does not activate CD8+ cytotoxic T cells (CTL) but when given in complete Freunds adjuvant (CFA), or in formulations of a number of novel adjuvants, an OVA‐specific CD8+ CTL response can be detected. We show in this report that immunization with soluble OVA mixed with heat‐killed Mycobacterium vaccae, but not with other common pathogenic and saprophytic mycobacteria, can activate OVA‐specific CD8+ CTL. An OVA‐specific CTL response is detected when mice are immunized by either the intraperitoneal or intranasal route and their spleen cells are re‐stimulated in vitro. Adjuvant activity of heat‐killed M. vaccae is present in M. vaccae culture filtrate, in soluble protein components of whole M. vaccae and in the 65 kDa heat‐shock protein (hsp) of M. vaccae. Mycobacterium vaccae has previously been shown to have no adverse side‐effects in humans. The current results suggest that M. vaccae may be useful as an adjuvant for vaccines and other immunotherapies where CD8+ CTL responses to exogenous proteins are crucial.

Dual fluorescence combined with a two-color immunoperoxidase technique: A new way of visualizing diverse neuronal elements

A method is described that allows an estimation of the neurotransmitter-related immunoreactivity, morphology and relationship to other immunoreactive elements, of single functionally identified neurons in the central nervous system. First, neurons are identified electrophysiologically using intracellular recording and labelled by iontophoresis of lucifer yellow (LY). After fixation and sectioning of the brain tissue, the location of the labelled neuron is determined by fluorescence microscopy. Sections are then processed using an indirect immunofluorescence procedure in order to determine the antigen content of the labelled neurons. Antisera to LY and an avidin-biotin immunoperoxidase technique is then used to localize LY in a permanent form, while the other previously localized antigen is permanently visualised by using the fluorescent-labelled second antibody as a bridge antibody in a peroxidase anti-peroxidase technique. The method is illustrated by an examination of neurons in the medulla oblongata of the rat, that have been stained intracellularly with LY, their content of tyrosine hydroxylase assessed, and their relationship to other tyrosine hydroxylase immunoreactive neurons determined.

Cytokine mRNA expressed in tuberculin skin test biopsies from BCG-vaccinated and Mycobacterium bovis inoculated cattle.

To obtain a better understanding of the delayed‐type hypersensitivity reaction to Mycobacterium bovis, we measured the expression of cytokine mRNA from tuberculin skin test biopsies of cattle. Non‐vaccinated and BCG‐vaccinated cattle were inoculated intratracheally with a low dose of virulent M. bovis or sham‐inoculated and 20 weeks later were skin tested with tuberculin. At necropsy 1–2 weeks later, tuberculous lesions were found in six of the nine non‐vaccinated and three of the nine BCG‐vaccinated animals. All of the lesioned and the majority of the non‐lesioned M. bovis inoculated cattle showed a distinct skin swelling response to tuberculin, irrespective of vaccination. However, cattle with tuberculous lesions displayed larger skin swelling responses than non‐lesioned cattle. Tuberculin‐induced expression of IFN‐γ, IL2, IL4, IL10 and TNF‐α mRNA occurred in the skin biopsies of all of the lesioned, M. bovis inoculated animals except for an absence of tuberculin‐induced TNF‐α mRNA expression in two animals. A lower proportion of the non‐lesioned M. bovis inoculated cattle displayed tuberculin‐induced expression of the five cytokine mRNA. There was no evidence of a unique pattern of cytokine expression which could be used to distinguish between diseased and protected animals. By 28 weeks after vaccination, the three BCG‐vaccinated, sham‐inoculated cattle displayed minimal skin swelling response to tuberculin, but tuberculin‐induced expression of IFN‐γ, IL2, IL4, TL10 and TNF‐α mRNA was observed in skin biopsies of all of these animals. The pattern of cytokine mRNA expressed in the tuberculin skin test reaction site of cattle appeared to be typical of a mixed T helper O‐like cell phenotype response and there was no clear‐cut relationship between the size of the tuberculin skin swelling response and cytokine mRNA expression.

Production of the 19-kDa antigen of Mycobacterium tuberculosis in Escherichia coli and its purification

The 19-kDa antigen (19Ag) of Mycobacterium tuberculosis (Mt) is a lipoprotein which is released from the organism during growth. In order to study the possible involvement of this antigen in the host protective response against Mt infection, it would be helpful to obtain high-level production of 19Ag from a recombinant organism. We have found that overexpression of the native 19Ag gene in Escherichia coli or yeast leads to products which are aggregated and insoluble. By site-directed mutagenesis of the 19Ag lipoprotein leader sequence, we have generated a mutant gene which directs the production of 19Ag into the periplasmic space of E. coli, from where it can be easily purified in high yield. 19Ag obtained from this mutant construct lacks the lipid-modified N-terminal Cys residue found in the native 19Ag, and is not glycosylated, but is otherwise indistinguishable from 19Ag isolated from Mt culture supernatant.

Expression of genes in cloned murine cell lines that can be maintained in both interleukin 2- and interleukin 3-dependent growth states.

Two cloned murine cell lines. FD.C/1 and 32Dcl‐23 exhibit switching of lymphokine‐dependent growth states. The bone marrow‐derived FD.C/1 and 32Dcl‐23 cell lines are normally grown in culture medium supplemented with inierleukin 3 (IL3). The replacement of IL3 with interleukin 2 (IL2) in the medium results in an increase in IL2 receptor expression in FD.C/1 and 32Dcl‐23 cells and the switching of cells lo an IL2‐dependent growth state. We have compared patterns of protein and phosphoprotein synthesis, as well as the expression of the c‐abl, c‐ras, c‐myb, and c‐fos oncogenes in these cell lines maintained in IL3‐ and I L2‐dependent growth states. The synthesis of a series of proteins and phosphoproteins are identified with each of the lymphokine‐dependent growth states. All of the oncogenes examined are expressed in both IL2‐ and lL3‐dependent cells and are not altered by phenotypic changes in lymphokine growth dependence. The relationship of oncogene expression to intracellular pathways regulated by lymphokine‐receptor interactions is considered.

Genetic control of immune responses to the 18-kDa protein of Mycobacterium leprae: Different TH1 subsets may be involved in proliferative and delayed-type hypersensitivity responses

In vitro and in vivo responses to the 18-kDa protein of Mycobacterium leprae have been analysed in different strains of mice. Lymphocytes from BALB/cJ (H-2d), BALB.B (H-2b), B10.BR (H-2k), and B10.M (H-2f) mice primed with 18-kDa protein yielded high T cell proliferative responses, while those from C57BL/10J (H-2b) mice yielded lower responses. Both H-2 and non-H-2 genes contributed to the magnitude of responsiveness. F1 mice from high and low responder strains showed high responsiveness to the 18-kDa protein. Supernatants from lymph node cell cultures prepared from 18-kDa protein-immunised BALB/cJ, B10.BR, and C57BL/10J mice contained IL-2 but no IL-4, indicating that activated T cells from both high and low responder mice were of a TH1 phenotype. Cell cultures from low responder C57BL/10J mice produced less IL-2 than those from high responders. The low responsiveness to the 18-kDa protein in proliferative assays might be due to a low frequency of antigen-specific T cells in the C57BL/10J mouse strain. BALB/cJ, C57BL/10J, and F1 (BALB/cJ x B10.BR) mouse strains were tested for in vivo DTH reactions to the 18-kDa protein. All strains, including C57BL/10J, were high DTH responders. Although DTH effector cells and 18-kDa protein-specific proliferative T cells belong to the TH1 subset, our data comparing high and low responder status indicate that distinct TH1 subpopulations are stimulated in response to the 18-kDa protein of M. leprae.

Expression of functional human interleukin-2 receptors in murine interleukin-3-dependent cells.

A murine recombinant retrovirus containing the cDNA encoding the human p55 interleukin‐2 (IL2)‐binding protein was used to insert this gene into a murine interleukin‐3 (IL3)‐dependent cell line, FD.C/1. Virus‐infected cells, maintained in medium supplemented with IL3, expressed human p55 on the cell surface and readily adapted to growth using human IL2. In the presence of human IL2, the synthesis of the endogenous murine p55 binding protein was induced in FD.C/1 cells, making it difficult to determine whether the human p55 protein was actively involved in the process of growth signal transduction. A cloned cell line, FD.huIL2R‐2, was identified which grew in the presence of human IL2 but which had lost the ability to synthesize murine p55 protein. Growth of this clone was inhibited by the monoclonal antibody 2A3 which specifically blocked binding of human IL2 to the human p55 binding protein. Analysis of restriction enzyme digests of FD.huIL2R‐2 cell DNA revealed that a rearrangement of a murine p55 gene had occurred, implying that virus infection had resulted in the integration of retroviral DNA at a site close to or within a murine p55 gene. IfIL2 signal transduction involves binding to a surface heterodimeric receptor for IL2, it is argued that FD.huIL2R‐2 cells contain an IL2 receptor complex of murine p70 and human p55 IL2‐binding proteins. Alternatively, it is possible that integration of human p55 DNA into a site close to a murine p55 gene may lead to a hybrid p55 IL2‐binding protein. If FD.huIL2R‐2 cells express murine p70 IL2‐binding protein as part of the receptor complex, the inability of cells to grow in murine IL2 implies that IL2 binding to p70 protein alone is insufficient for a growth signal in these cells. FD.huIL2R‐2 cells grow at rates similar in IL3‐ or human IL2‐dependent states. It is likely therefore that the biochemical pathways that control each of these lymphokine‐dependent growth states are very similar.

Chapter 6 Biochemical Applications of Preparative Liquid Chromatography

Publisher Summary This chapter discusses the biochemical applications of preparative liquid chromatography (LC). While the preparative separation of amino acids and small peptides by reversed phase LC is becoming an accepted procedure, the separation of proteins by this technique still requires careful evaluation. The biological activities of many proteins are sensitive to denaturation by extremes in pH, by contact with organic solvents or high salt concentrations, by adsorption onto glass or hydrophobic moieties, or at an air–water interface. Therefore a judicious choice of reversed phase columns with a suitable choice of organic modifier and optimal ionic modifier concentrations has allowed the separation of proteins with retention of biological activity. For the peptide separations, the addition of an ionic modifier is often essential for the separation of protein samples by reversed phase LC. The addition of ionic modifiers to the mobile phase can also give useful differences in the resolution of complex protein mixtures. The ionic modifiers most widely used include acids such as trifluoroacetic, phosphoric, and hydrochloric; and the salts of organic bases, such as triethylamine phosphate and pyridine formate.