Of increasing concern among higher education institutions is the problem of maintaining the quality of education in the face of increased student numbers and continuing funding cuts. These concerns, coupled with the advent of readily accessible and relatively cost effective computer technology, has seen a marked increase in the use of computer-based education delivery systems in universities. Much debate now centres on how effective and beneficial computer-based learning (CBL) is, particularly with respect to learning and cognitive processes (Brown et al. 1989; Rowe 1993), student performance (Mevarech et al. 1991; Stewell and Delpierre 1992; Blackmore and Britt 1993; Mevarech 1993; Chambers et al. 1995), gender and age related performance (Massoud 1991; Lee 1993; Colley et al. 1994; Dyck and Smither 1994) and software design and mode of delivery (Ahern 1993; Jackson et al. 1993; Rowe 1993; Chambers et al. 1995). Addressing these questions is difficult due to a lack of published data and because comparisons of student performance are difficult given the highly diverse ways in which CBL is approached, implemented and evaluated. One area to which CBL readily lends itself is the use of computer simulations in practical or laboratory teaching. Laboratory classes aim to teach students principles of experimental design, data collection, data processing as well as the correct usage of commonly used laboratory equipment. The application of computer simulations can be beneficial if (i) the costs of running a particular experiment are prohibitive (ii) time constraints apply e.g., growth or breeding experiments (iii) ethical or humane considerations are involved e.g., experiments involving euthanasia of large numbers of animals (iv) it is difficult to observe or manipulate the phenomenon under consideration e.g., nutrient cycling in a plant community (v) it is important that students are familiar with a piece of equipment or procedure prior to undertaking an experiment e.g., familiarity with the use of a spectrophotometer (vi) rapid and efficient feedback on a students understanding of the subject material is desirable (vii) the experiments are technically demanding (ix) require the use of dangerous or radioactive compounds. This talk will be primarily on the use of computer simulations in biology but I will talk about laboratory marking programs and tutorial assessment programs.

Betacellulin (BTC) belongs to the epidermal growth factor (EGF) family of peptide ligands that are characterized by a six-cysteine consensus motif (EGF-motif) that forms three intra-molecular disulfide bonds, crucial for binding the ErbB receptor family. A variety of in vitro studies have identified BTC as an important factor in the growth and/or differentiation of pancreatic islet cells. The molecular mechanisms that regulate the transcription of the BTC gene however have not been delineated. As an initial step, we have characterized the genomic structure of the mouse BTC (mBTC) gene. mBTC cDNA was used as a probe to screen a mouse 129/SVJ genomic bacterial artificial chromosome (BAC) library. Three positive clones containing the entire gene were isolated. DNA sequence analysis identified six exons (1–6) and five introns (A–E); a structure conserved among the EGF family. PCR analysis showed that introns A–E are approximately 7.8, 8.9, 3.8, 1.4 and 1.4 kb in length, respectively. The EGF-motif is encoded by exons 3 and 4 with an intron (intron C) disrupting the coding sequence between the second and third disulfide loops. All exon–intron boundaries are consistent with the “gt-ag” rule. Multiple transcription start sites and one poly(A) site, located 18 bp downstream of a polyadenylation signal sequence, were identified by 5′- and 3′-RACE, respectively. Approximately 2.6 kb of 5′-flanking region was sequenced and was shown to lack consensus TATA and CCAAT boxes, but was found to contain several putative cis-acting regulatory elements. These included consensus binding sites for transcription factors HNF3β, USF, Nkx2–5, AP-4, and Sp1. Functional promoter analysis of the 5′-flanking region in COS-7 cells, using 5′-deletion fragments (−168/+335; −635/+335; −732/+335; −1175/+335; −1698/+335) cloned into a promoterless firefly luciferase reporter vector, identified basal promoter activity and both positive and negative cis-acting elements.

Growth factors and their binding proteins are important proteins regulating mammalian cell proliferation and differentiation so there is considerable interest in producing them as recombinant proteins, especially in hosts that do not already produce a complex mixture of growth factors. Many growth factors require posttranslational modifications making them unsuitable for production in Escherichia coli or other prokaryotes. Since several expression vector systems have been recently developed for foreign protein production in the cellular slime mould, Dictyostelium discoideum, we attempted to use two of these systems to express human insulin-like growth factor binding protein 6 (hIGFBP6) and bovine beta-cellulin (bBTC) as secreted proteins. Although both proteins were successfully produced in stably transformed amoebae, no secretion was detected in spite of several attempts to facilitate this occurring.

The sori of Dictyostelium discoideum (strains SG1, SG2, NC4 and V12) contained more than 100 mM ammonium phosphate. Glutamine synthetase (GS), which could remove ammonia from the sorus, was not present in 2-d-old dormant spores but enzyme activity returned to vegetative levels after spore germination. Based on mRNA blotting, the activity of this enzyme in germinating spores appeared to be transcriptionally controlled. At the same time that GS activity was increasing, ammonia was released from germinating spores. Exogenous ammonium ions at a concentration of 28 mM did not block germination nor modulate GS activity in nascent amoebae. It was concluded that the transcription and translation of GS is not environmentally regulated but is an integral part of the germination process, preparing nascent amoebae for vegetative growth. An exogenous concentration of 69 mM ammonium phosphate could maintain dormancy in spores of strains SG1 and SG2 for at least a week in the absence of any other inhibitory component from the sori. The inhibition was reversible at any time either by dilution or by washing the spores free of the ammonium ion. Spores of strain acg- were not inhibited by 100 mM ammonium phosphate. A model is presented in which GS in prespore cells serves as a sink for ammonia to allow the osmotically sensitive adenylyl cyclase aggregation protein (ACA) to activate protein kinase A (PKA) to induce fruiting-body formation. After fruiting-body formation is complete, the decline in GS and ACA activities in developing spores is offset by their replacement with the osmotically and ammonia-stimulated adenylyl cyclase osmosensor for germination (ACG). Ammonia and discadenine may act as separate signals to synergistically activate PKA by stimulating ACG activity while inhibiting cAMP phosphodiestrase activity in fully dormant spores.

Kangaroo IGF-II has been purified from western grey kangaroo (Macropus fuliginosus) serum and characterised in a number of in vitro assays. In addition, the complete cDNA sequence of mature IGF-II has been obtained by reverse-transcription polymerase chain reaction. Comparison of the kangaroo IGF-II cDNA sequence with known IGF-II sequences from other species revealed that it is very similar to the human variant, [Ser(29)]-hIGF-II. Both the variant and kangaroo IGF-II contain an insert of nine nucleotides that encode the amino acids Leu-Pro-Gly at the junction of the B and C domains of the mature protein. The deduced kangaroo IGF-II protein sequence also contains three other amino acid changes that are not observed in human IGF-II. These amino acid differences share similarities with the changes described in many of the IGF-IIs reported for non-mammalian species. Characterisation of human IGF-II, kangaroo IGF-II, chicken IGF-II and [Ser(29)]-hIGF-II in a number of in vitro assays revealed that all four proteins are functionally very similar. No significant differences were observed in the ability of the IGF-IIs to bind to the bovine IGF-II/cation-independent mannose 6-phosphate receptor or to stimulate protein synthesis in rat L6 myoblasts. However, differences were observed in their abilities to bind to IGF-binding proteins (IGFBPs) present in human serum. Kangaroo, chicken and [Ser(29)]-hIGF-II had lower apparent affinities for human IGFBPs than did human IGF-II. Thus, it appears that the major circulating form of IGF-II in the kangaroo and a minor form of IGF-II found in human serum are structurally and functionally very similar. This suggests that the splice site that generates both the variant and major form of human IGF-II must have evolved after the divergence of marsupials from placental mammals.

The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993) Endocrinology 133, 440-446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195-204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage.

Insulin-like growth factor-I (IGF-I) and IGF-II have been purified to homogeneity from kangaroo (Macropus fuliginosus) serum, thus this represents the first report of the purification, sequencing and characterisation of marsupial IGFs. N-Terminal protein sequencing reveals that there are six amino acid differences between kangaroo and human IGF-I. Kangaroo IGF-II has been partially sequenced and no differences were found between human and kangaroo IGF-II in the 53 residues identified. Thus the IGFs appear to be remarkably structurally conserved during mammalian radiation. In addition, in vitro characterisation of kangaroo IGF-I demonstrated that the functional properties of human, kangaroo and chicken IGF-I are very similar. In an assay measuring the ability of the proteins to stimulate protein synthesis in rat L6 myoblasts, all IGF-I proteins were found to be equally potent. The ability of all three proteins to compete for binding with radiolabelled human IGF-I to type-1 IGF receptors in L6 myoblasts and in Sminthopsis crassicaudata transformed lung fibroblasts, a marsupial cell line, was comparable. Furthermore, kangaroo and human IGF-I react equally in a human IGF-I RIA using a human reference standard, radiolabelled human IGF-I and a polyclonal antibody raised against recombinant human IGF-I. This study indicates that not only is the primary structure of eutherian and metatherian IGF-I conserved, but also the proteins appear to be functionally similar.

Glutamine synthetase (GS) from the cellular slime mould Dictyostelium discoideum was purified to apparent electrophoretic homogeneity with a final yield of 21.7%. The native enzyme appeared to be a GS-II type enzyme. SDS-PAGE of the final preparation revealed a single band of 43.5 kDa. The enzyme has a native molecular mass of 376 kDa, determined using Superose 6, indicating that the enzyme is likely to be an octamer of identical subunits. Dictyostelium discoideum GS has an optimal temperature of 42.5 degrees C, although it is thermolabile in the absence of L-glutamate and (or) Mg2+-ATP. The enzyme exhibits a K-m for L-glutamate, ATP, and NH4Cl of 2.18, 0.18, and 0.11 mM, respectively, in the L-glutamine synthetic reaction with an optimal pH of 7.9. GS from D. discoideum does not appear to be significantly inhibited by various end products of L-glutamine metabolism, although it is potently inhibited by methionine sulphoximine. These properties are those expected for an enzyme for which the primary function is the assimilation of ammonia.

To examine further the role of the enzyme glutamine synthetase in Dictyostelium discoideum we report here the effects of a specific glutamine synthetase inhibitor, methionine sulfoximine, on the growth and differentiation of this organism. Vegetative AX3 cells grown in the presence of methionine sulfoximine did not complete culmination in the normal time but were blocked at the finger stage. In these cells glutamine synthetase activity was almost completely abolished. However, methionine sulfoximine did not affect the level of glutamine synthetase mRNA, suggesting that there is no link between glutamine synthetase activity and mRNA transcription. Eventually glutamine synthetase activity reappeared and at the time culmination occurred. These results suggest that glutamine synthetase plays an important role in the assimilation of ammonia during the later stages of development in D. discoideum and that this assimilation is necessary for the completion of culmination.

The enzyme glutamine synthetase (GS) of Dictyostelium discoideum is developmentally regulated, preferentially localized in prespore cells and is likely to play an important role in controlling the levels of ammonia, a known morphogen, in this organism. To further investigate the regulation of GS, a portion of the GS gene was isolated and used as a probe to examine the changes in GS mRNA throughout development and the level of GS mRNA in the two precursor cell types. The amino acid sequence of the cloned DNA fragment isolated is highly homologous to other eukaryotic GS genes. DNA blot analysis demonstrated that the GS gene exists as a single copy in D. discoideum . Analysis of RNA indicated that there is a single 1.7 kb GS transcript that increased during development to peak at the initial stages of culmination. Furthermore, GS mRNA was highly localized in prespore cells, which is consistent with a proposed source–sink model for ammonia assimilation in this organism.

Two forms of the NAD-dependent glutamate dehydrogenase were partially purified from Dictyostelium discoideum, an activated and a non-activated form. V(max) for the non-activated enzyme was stimulated 88-fold and the activated enzyme 3-fold by 0.1 mM AMP (at their pH optima). Half maximal stimulation by AMP is achieved at 221 +/- 39 microM for the non-activated enzyme and 20 +/- 2 microM for the activated enzyme. We have shown that activation of NAD-GDH in vivo has many similarities to trypsin treatment of non-activated enzyme and that proteolysis is the probable mechanism of activation.

The enzyme glutamine synthetase (GS) is described for the first time in Dictyostelium discoideum. The appearance of this enzyme is developmentally regulated. The level of activity is low in vegetative cells and increases more than threefold during differentiation. Furthermore this enzyme is shown to be differentially localized in prespore cells, the specific activity being approximately fourfold higher than in prestalk cells. The enzyme has a pH optimum of 7.8 and 8.2 in the gamma-glutamyltransferase and gamma-glutamylsynthetase assays, respectively, and a temperature optimum of 45 degrees C. Kinetic studies of GS revealed apparent Km values of 5.9 mM, 0.009 mM and 8.6 mM for glutamine, ADP and NH2OH, respectively, in the gamma-glutamyltransferase assay, and of 2.2 mM, 0.12 mM and 0.64 mM for glutamate, ATP and NH2OH, respectively, in the gamma-glutamylsynthetase assay.

A protein kinase from Dictyostelium discoideum which phosphorylates the synthetic peptide, calmodulin-dependent protein kinase substrate (CDPKS, amino acid sequence: PLRRTLSVAA) and is stimulated by Ca2+/calmodulin is described. This is the first report of a protein kinase with these characteristics in D. discoideum. The enzyme was partially purified by Q-Sepharose chromatography. The protein kinase is very labile, and rapidly loses Ca2+/calmodulin-dependence upon standing at 4 degrees C, even in the presence of protease inhibitors, making further purification and characterisation difficult. In the active fractions, a 55 kDa polypeptide is labelled with [gamma-32P]ATP in vitro under conditions in which intramolecular rather than intermolecular reactions are favoured. The phosphorylation of this peptide is stimulated in the presence of Ca2+ and calmodulin but not Ca2+ alone. Ca2+/calmodulin-dependent stimulation is inhibited in the presence of the calmodulin antagonist, trifluoperazine (TFP). It is proposed that the 55 kDa polypeptide may represent the autophosphorylated form of the enzyme.

Incubation of Dictyostelium discoideum cells with selenate is known to inhibit vegetative growth. In this paper we show that in the presence of selenate macromolecules accumulate which can be converted to sulphated products once the selenate is removed. The presence of cycloheximide, an inhibitor of protein synthesis, during the subsequent incubation does not prevent this conversion but tunicamycin, an inhibitor of glycosylation does. It is concluded that, in the presence of selenate, precursors accumulate as unglycosylated proteins, suggesting that feedback inhibition of glycosylation may be operated.

In extracts from vegetative Dictyostelium discoideum V12 the basal NAD-dependent glutamate dehydrogenase (NAD-GDH) activity was low, but it increased on standing at 4 degrees C. When 0.1 mM-AMP was included in the assay mix, enzyme activity was stimulated nearly 30-fold. As the extract was allowed to age, the enzyme rapidly lost its ability to be stimulated by AMP. The response of NAD-GDH to AMP was also dependent on the stage of morphogenesis. The ratios of NAD-GDH activity assayed with and without AMP (+AMP/-AMP ratios) in freshly prepared extracts from cells at 0, 4, 8 and 12 h of development were similar, but declined later in morphogenesis. The +AMP/-AMP ratio decreased sharply during activation at 4 degrees C in extracts from cells at 0, 4, 16 and 20 h of development. By contrast, extracts from cells starved for 8 and 12 h remained more responsive to AMP throughout activation. Analysis of Western blots showed that vegetative NAD-GDH did not undergo any detectable proteolytic cleavage during 96 h of activation at 4 degrees C. Also, no change in molecular mass appeared to take place within the cells until culmination (20-24 h), when some breakdown products appeared. Activation of NAD-GDH also occurred in D. discoideum strains NC4 and AX3, and in D. mucoroides. In addition, the enzyme from these four strains was stimulated by AMP and the +AMP/-AMP ratio declined with similar kinetics during activation. The enzyme from Polysphondylium violaceum was not activated on standing, but it was stimulated by AMP. The effect of activation of NAD-GDH is discussed in relation to a postulated catabolic role for this enzyme.

The NAD-dependent glutamate dehydrogenase (GDH) from Dictyostelium discoideum was purified 1101-fold with a yield of 23.4%. The enzyme has an apparent Mr of 356 kDa, determined using Sephacryl S400, and a subunit molecular weight of 54 kDa on SDS-polyacrylamide gel electrophoresis. The Kms for alpha-ketoglutarate, NADH, and NH4+ are 0.36 +/- 0.03 mM, 16.0 +/- 0.1 microM, and 34.5 +/- 2.7 mM, respectively. The purified enzyme has a pH optimum of pH 7.25-7.5. At 0.1 mM, ADP and AMP stimulate GDH activity 25 and 102%, respectively. Half-maximal activity in the presence of 0.1 mM AMP for alpha-ketoglutarate, NADH, and NH4+ is reached at 2.3 +/- 0.1 mM, 71.4 +/- 5.5 microM, and 27.9 +/- 3.6 mM, respectively.

Pyruvate kinase (EC 2.7.1.40) from aggregating Dictyostelium discoideum cells has been purified to homogeneity. It has a monomeric molecular weight of 66kD and is tetrameric in low ionic strength buffers. The enzyme is not regulated by fructose 1,6-bisphosphate or by alanine and appears to resemble the M1 isoenzyme from rat liver most closely, although its activity is not inhibited by ATP.