Membrane dipeptidase (EC 3.4.13.19) is a glycosylphosphatidylinositol-anchored glycoprotein of the renal brush border which exists as a disulfide-linked homodimer. Porcine membrane dipeptidase has a subunit M(r) of 47 kDa, and the mature protein contains seven cysteine residues per subunit, six of which are conserved in the human enzyme. Chemical modification established that cysteine residues are not involved in enzyme activity. In order to determine which of the cysteine residues are involved in the interchain disulfide bond, we have used a site-directed mutagenesis approach. Each of the conserved cysteine residues was replaced by glycine or alanine. The single mutants (C71G, C93A, C154G, C226A, C258G, and C361G) were expressed in COS-1 cells and their enzymatic activity and oligomeric structure determined. Only the C361G mutant migrated as a polypeptide of 47 kDa when subjected to denaturing polyacrylamide gel electrophoresis under nonreducing conditions. Thus, cysteine 361 is the only residue involved in disulfide linkage between the subunits. This places the disulfide bond close to the site of GPI anchor addition (Ser 368 in the porcine enzyme) and to the membrane surface. Titration of the human and porcine proteins with 2-nitro-5-thiosulfabenzoate indicates that membrane dipeptidase additionally possesses two intrachain disulfide bonds. On native polyacrylamide gel electrophoresis, the C361G mutant migrates in a manner identical to that of the wild type, indicating that the protein remains associated as a noncovalent homodimer. The expressed C361G mutant, unlike the wild type, is released from COS-1 cell membranes by trypsin and by an endogenous serine protease.

Aminopeptidase P (APP, EC 3.4.1 1.9) was first identified in n exopcptidase that releases the N-terminal amino acid from peptides with a proline in the second position [ I ] . APP activity has been shown in a range of organisms and an APP from E.co/i has been cloned and sequenced [2]. APP was first purified to homogeneity from pig kidney microvillar membranes [3J and has been shown to be anchored to the membranes by a glycosyl-phosphatidylinositol (GPI) moiety [4].In the original purification of APP from pig kidney [ 3 ] the enzyme was released from the microvillar membranes by treatment with bacterial phospholipase C (PLC). The solubilised enzyme was then purified to homogeneity in a series of chromatographic steps 131. Enzyme activity was monitored throughout by HPLC using a synthetic peptide, Gly-Pro-HyPro, as substrate [4]. Purified APP was used to raise a polyclonal antibody (RP171) in rabbits. RP171 also recognised the crossreacting determinant of PLC-cleaved GPI-anchored proteins [5]. Antibodies recognising the anchor epitope were removed by affinity chromatography with PLC-cleaved membrane dipeptidase used as a ligand. The resultant polyclonal, RP172, recognised a single polypeptide of 91 kDa in a Western blot of PLC-solubilised microvillar membranes.In order to reduce the time of purification and increase the yield of APP in purification, RP172 was coupled to cyanogen bromide-activated Sepharose and used to purify kidney APP by immunoaffinity chromatography. Following APP solubilisation from pig kidney microvillar membranes by l3.cereu.s PLC (0.35 unitdmg in lOmM Tris-HCI, 0.1M NaCl, pH 7.4 at 37oC for 2h) 60% of the APP activity was solubilised. The solubilised fraction was dialysed extensively against phosphatt-buffered saline before being applied to the RP172 affinity column. Purified APP was eluted with 0. IM ethanolamine, pH1 1 and showed a 200-fold enrichment over kidney cortex homogenate.Attempts at N-terminal sequencing of pig kidney APP have revealed that the enzyme is N-terminally blocked. Following proteolytic cleavage of APP with endoproteinases GluC, ArgC and LysC, peptide sequence data for the enzyme have been obtained.Three degenerate oligonucleotides (26-mer, 64 degeneracy, 23-mer. 256 degeneracy, 17-mer, 768 degeneracy) primers have been designed, synthesised and used in screening of a pig kidney cDNA library. Several positive clones have been identified and are being further characterised.APP activity was observed in pig intestine and, to characterise this activity further, brush border membranes were prepared from pig small intestine [ 6 ] . The specific activity in these membranes was approx. 30nmol Pro-HyProlmidmg as compared to a specific acitivity of approx. 55nmol ProHyPro/midmg in pig kidney microvillar Inembrdnes (as assayed by HPLC). Western blot analysis of pig intestinal brush border membranes with RP172 identified two proteins of 95 kDa and 105 kDa, in contrast to the single protein identified in kidney microvillar membranes of 91 kDa. Incubation of brush border membranes w...

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Since email access has become almost universal, Universities have increasingly used email as a key communication channel. This project investigated the number and origin of email communications to students on three Open University first year undergraduate STEM (Science, Technology, Engineering and Mathematics) modules. The modules were: Topics in Science (S142), Introducing Health Sciences (SDK125) and Environment: Journeys through a changing World (U116). S142 and SDK125 are 30 credit modules amounting to about 8 hours study a week and U116 is 60 credits amounting to around 16 hours study per week and all three modules are run over 31 weeks. The number of students who started studying the modules in February 2015 was; S142 838, SDK125, 824 and U116, 494 which ran from February to October presentation in 2015.Quantitative analysis of the number and type of emails sent to students on these modules revealed an average and maximum number of email communications per student on each module was S142: average 38, maximum 59, SDK125: average 67, maximum 82, U116: average 45, maximum 83. These figures are for students studying a single module over 31 weeks.Qualitative analysis from interviews undertaken with 40 students from each module determined how they felt about the number and type of communications they were receiving. The outcome of this analysis revealed that students appreciated email as the main form of communication from the university, although they underestimated quite significantly the number of emails they had received.Keywords: Student experience, email communications, STEM, distance learners. PROJECT RATIONALEThe increasing penetration of the Internet across the World over the last few years and increased use of the internet has allowed growth of online courses and subsequent increased use of email as a form of communication with students. In 2011-12 116,535 international students were enrolled in some form of distance, flexible or distributed learning with a UK institution of higher education [1]. Consequently institutions have needed to adapt their communication and student support to build relationships with these remote students.The aim of this project was to investigate the perception in the Open University (OU) that students may be receiving too many email communications from the university, which could potentially lead to the students feeling overwhelmed and confused. The Open University supports over 170,000 distance learning students each year across a range of over 450 undergraduate, postgraduate and professional modules. Teaching is delivered through module materials delivered both online and in hard copy and academic support is provided by tutors and faculty staff at a distance using electronic and telephone communications.The literature is inconclusive regarding optimal levels of contact to manage email based remote relationships. In a commercial context, Kim et al [2] report that customers usually want less frequent Customer Relationship Management (CRM) contact than marketers think...