Phosphorylation of eukaryotic translation initiation factor 2 (eIF2) is one of the best studied and most widely used means for regulating protein synthesis activity in eukaryotic cells. Control through eIF2 is exerted by its phosphorylation, which disrupts the guanidine exchange cycle that is required for every initiation event, and thereby inhibits translation. The eIF2 pathway regulates protein synthesis in response to stresses, viral infections, and nutrient depletion, among others. We present analyses of an ordinary differential equation-based model of this pathway, which aim to identify its principal robustness and stability conferring features using linear control theory. The eIF2 pathway can respond sensitively, appropriately and in a timely fashion to some changes in the environment of the cell, while being robust and unresponsive to other types of change. Neither the way in which appropriate responses are achieved (responsiveness), nor how inappropriate responses are avoided (robustness), is currently well understood. Our analyses indicate that, within eIF2 dependent regulatory model the robustness do not arising from the properties of any one individual pathway species rather is a distributed property. On the other hand, stability lies in the structure of the model that is damaging the structure produces major alterations in the stability. Further it is observed that, key non-linearities within the system helps in maintaining transient behaviour and removal or linearisation of key non-linearities can generate undesirable results such as negative cellular concentration. Our analyses also indicate existence of natural sliding surface within the eIF2 dependent regulatory system that helps the system in counteracting uncertainties lies in the input channel. Further, the role of uncharged transfer ribonucleic acid (tRNA) and protein kinase R (PKR) signalling on general translation rate as well as on phosporylation of eIF2-alpha is also investigated by extrapolating the proposed computational yeast model to the case of mammalian cells. It is observed that PKR is compensating the loss of general control nonderepressible 2 (GCN2) and maintaining levels of phosphorylated eIF2-alpha in tumours, while signalling strength of uncharged tRNA is responsible for delivering statistically significant difference in the ratio of phosphorylated eIF2 and alpha-subunit of eIF2 for mixed background and C57BL6 sarcomas.

The receptor-type protein tyrosine phosphatases (RPTPs) of the R3subgroup play key roles in the immune, vascular and nervous system. They are characterised by an extracellular domain (ECD), comprised of multiple FNIII-like repeats, a transmembrane domain and a single intracellular phosphatase domain. Although their phosphatase domains have been Fstudied in detail the functional roles of their extracellular regions have not been clearly defined. Potential roles in ligand interaction, dimerisation and cell-cell contacts have been reported. Here I used a bimolecular fluorescence complementation (BiFC) assay in live cells to examine the molecular basis for the interaction of one of the R3 RPTP members, VE-PTP, with VE-cadherin, and explored the potential of others to interact with this protein. The potential of R3 RPTPs to homo-dimerise via extracellular domains in live cells was also addressed. Quantitative BiFC analysis using sialophorin (SPN), an unrelated membrane protein, and a membrane anchored C-terminal Venus-YFP (Myr-VC) fragment as controls revealed a specific interaction between VE-PTP and VE-cadherin using constructs expressing only the extracellular and transmembrane domains. Use of a deletion mutant indicated that, in contrast to previous studies, removal of the 17th FNIII-like domain of VE-PTP is not sufficient to disrupt this interaction. Other members of the R3 RPTP family (DEP-1, GLEPP1 and SAP-1) also exhibited the potential to interact with VE-cadherin suggesting that specificity of this protein-protein interaction is not determined by the ECD alone. The direct interaction of DEP-1 with VE-cadherin is likely to be of physiological relevance since both proteins are expressed in endothelial cells. GLEPP1 and SAP-1 exhibited homo-dimerisation, whereas DEP-1 and VE-PTP did not form dimers via their extracellular and/or transmembrane domains. SPN was identified as a possible bona fide ligand for DEP-1 and their interaction is likely to be of physiological relevance since they were both shown to regulate T cell receptor activation. The interactions identified in the present study suggest a role for both the extracellular domain and transmembrane domain of R3-PTPs in interaction with VE-cadherin. The study also highlights the importance of using multiple controls in BiFC experiments and quantitative analysis of results.

The amino acid compositions of rabbit IgG and its constituent polypeptide chains, produced by reduction and papain cleavage, have been determined, and these have been shown to be consistent with a four chain structure for IgG. Digestion of heavy chain with Pronase and isolation of those peptides lacking a free G-amino group has shown that pyrrolid-2-one-5-carboxylic acid is N-terminal in all molecules but that the sequence following this residue is mixed. The sequences present in pooled rabbit IgG were in the following approximate proportions: PCA-Ser-Val-Glu, 50%; PCA-Ser-Leu-Glu, 20%; PCA-Glu NH2, 20%. The heavy chains of a purified antibody, anti-(human serum albumin), and of IgG from a rabbit homozygous with respect to allotype (Aal/Ab5) both showed a similar mixed N-terminal sequence. Isolation of peptides from a tryptic digest of the Fd fragment has led to the extension of the first two sequences to nine residues. These were shown to be: PCA-Ser-Val/Leu-Glu-Glu-Ser-Gly-Gly-Arg. The yields of other tryptic peptides isolated lend some support to the concept of 'common' and 'variable' regions of sequence in Fd. The Fd fragment has also been studied by cleavage with cyanogen bromide and the results suggest that there are three methionine residues which are all present in fractional amounts in Fd prepared from pooled rabbit serum. One of these methionines has been shown to be present in 35-40% of the molecules at a position 35 residues from the N-terminal end. The sequence of the fragment obtained by cleavage at this methionine has been determined, and shown to be an extension of the N-terminal tryptic nonapeptide sequence. The relationship of this sequence to that of a similar fragment isolated from the heavy chain of human IgG is discussed.

The purpose of this work has been to correlate the level of certain enzymes in blood serums with clinical status. The introduction is concerned with the historical background of clinical enzymology and the general conditions which cause alterations in the level of enzymes in serum. Reasons are given for studying the seven selected enzymes, acid and alkaline phosphates. Appendix. Biochemical and case histories and detailed results of the enzyme determinations on the individual cancer patients are given in this section.

The outward movement of sodium was studied in isolated diaphragm muscle of the rat and in a toe muscle (flexor digitorum brevis TV) of the mouse by means of the isotope ²⁴Na. The sodium exchanged in rat diaphragm with a half-time of approximately five minutes, and the fibre sodium was estimated by compartmental analysis. Strophanthin (10-⁴g/m1) slowed the outward movement of sodium in rat diaphragm and caused an increase in the sodium content of the muscle and a decrease in its potassium content. It was found that these effects were reversible and it was therefore possible during the recovery to demonstrate net extrusion of sodium from the diaphragm. Strophanthin also slowed the outward movement of sodium in mouse muscle and increased its sodium content while the potassium content was decreased. Insulin (0.1 unit/ml.) increased the rate of outward movement of sodium in mouse muscle. It was found that the exchange of sodium in diaphragm muscle which had been denervated (8 days) was more rapid, the half-time being 4.1 min. for denervated muscles as compared with 5.4 min. for controls. The experiments were conducted on a factorial design based on a 4.point assay, and these differences are significant (P<0.01).

No description available.

No description available.

Artificial proteins and enzymes have the potential to aid in the production of pharmaceuticals and to facilitate basic biomedical research. Two methods currently exist for the development of artificial proteins: rational design and de novo selection. Rational design requires detailed knowledge of enzyme catalysis in order to design an enzyme active site in silico, and then introduce this active site into a protein. However, gaps in the understanding of protein folding and structure-function relationships make this approach challenging and far from routine. In contrast, laboratory evolution approaches to isolate artificial proteins and enzymes from libraries of variants are well established. In vitro selection techniques are powerful tools for the exploration of large areas of sequence space (up to 1013 unique sequences) in the search for functional proteins and enzymes. mRNA display selection methods have only recently been developed, and the application of this technique for the engineering of de novo enzymes has not been fully explored. This thesis describes the establishment of an mRNA display platform for the selection and evolution of novel proteins and enzymes from large, high-diversity libraries. The synthesis of novel selection substrates are described that will facilitate the application of mRNA display to the selection of Diels-Alderase enzymes. A novel application of mRNA display is described for the solution-phase selection of protein-ligand pairs using interaction-dependent reverse transcription. Further development of this research could increase the throughput of ligand discovery to complement the pace at which new macromolecular targets of interest are being discovered. The ability to generate tailor-made enzymes that catalyse novel reactions is of considerable interest. The applications of mRNA display selection described in this thesis will help to extend the range of enzyme catalysis, and to elucidate basic mechanisms of biocatalysis and protein evolution. Moreover, such ‘designer enzymes’ hold promise for a huge range of applications including, but not limited to, the synthesis of chemicals, pharmaceuticals, and production of renewable fuels.

VSV-G has been used for several years to pseudotype reteroviral and lentiviral vectors to increase the range of cell types that these vectors can be targeted to as well as increasing transfection efficiency and serum resistance. It has previously been shown that purified VSV-G protein can be added to several types of non-viral complexes to produce these same advantages. VSV-G therefore holds great potential in gene therapy for both viral and non-viral vectors. Due to the cellular toxicity of VSV-G in mammalian cells VSV-G pseudotyped viral vectors are generally produced from transiently transfected cells which greatly limit the scale of viral production. VSV-G for non-viral vectors is also limited in the same manner but also suffer from expensive and time consuming methods to purify the VSV-G from the expression media. To address these problems with production we attempted to generate strains of the yeast Saccharomyces cerevisiae that can produce VSV-G pseudotyped lentivirus and VSV-G protein from inducible integrated vectors. We theorised that the cell wall of Saccharomyces cerevisiae would prevent syncytia and cellular toxicity of VSV-G during production, allowing the continuous production of virus or protein. In this report we show that this new production method allows us to produce and purify VSV-G from yeast using simple and scalable methods and that this produces a greater enhancement of transfection efficiency than mammalian derived VSV-G. However we were not able to demonstrate the production of VSV-G pseudotyped virus, seemingly due to the genotoxic effects of viral integrase.

No description available.