Timothy M. Harrison

Crystal structure of the platelet glycoprotein Ib(alpha) N-terminal domain reveals an unmasking mechanism for receptor activation.

Glycoprotein Ib (GPIb) is a platelet receptor with a critical role in mediating the arrest of platelets at sites of vascular damage. GPIb binds to the A1 domain of von Willebrand factor (vWF-A1) at high blood shear, initiating platelet adhesion and contributing to the formation of a thrombus. To investigate the molecular basis of GPIb regulation and ligand binding, we have determined the structure of the N-terminal domain of the GPIbα chain (residues 1–279). This structure is the first determined from the cell adhesion/signaling class of leucine-rich repeat (LRR) proteins and reveals the topology of the characteristic disulfide-bonded flanking regions. The fold consists of an N-terminal β-hairpin, eight leucine-rich repeats, a disulfide-bonded loop, and a C-terminal anionic region. The structure also demonstrates a novel LRR motif in the form of an M-shaped arrangement of three tandem β-turns. Negatively charged binding surfaces on the LRR concave face and anionic region indicate two-step binding kinetics to vWF-A1, which can be regulated by an unmasking mechanism involving conformational change of a key loop. Using molecular docking of the GPIb and vWF-A1 crystal structures, we were also able to model the GPIb·vWF-A1 complex.

NOVEL MARKERS FOR CONSTITUTIVE SECRETION USED TO SHOW THAT TISSUE PLASMINOGEN ACTIVATOR IS SORTED TO THE REGULATED PATHWAY IN TRANSFECTED PC12 CELLS

The rat pheochromocytoma cell line PC12 contains two distinct pathways of protein secretion. Proteins secreted via the regulated pathway are stored in secretory vesicles and exocytosed only in response to a specific signal, whereas proteins secreted via the constitutive pathway are exported continuously. Analysis of regulated secretion of a heterologous protein in this system often relies on comparison of secretion rates with those of endogenous proteins known to be secreted via the constitutive route. In order to improve these controls, we have evaluated a number of secreted enzymes, selected for the sensitivity and convenience of their assays, as transgenic markers for the constitutive pathway. We show that both human‐secreted placental alkaline phosphatase (SEAP) and bacterial β‐lactamase operate in this way in transfected PC12 cells. In contrast, transfected human tissue plasminogen activator (tPA) is shown to be sorted to the regulated pathway.

Monoclonal antibodies against the 1,4-dihydropyridine receptor associated with voltage-sensitive Ca2+ channels detect similar polypeptides from a variety of tissues and species

Four monoclonal antibodies have been raised against voltage‐sensitive Ca2+ channel dihydropyridine receptors from rabbit skeletal muscle. When tested by immunoblot assay of denatured transverse tubule membranes in reducing polyacrylamide gels, each recognised a single polypeptide of Mr ~ 140000 that co‐migrated with the large glycoprotein subunit of the purified receptor. In blots of nonreducing gels, a larger protein of Mr ~ 170000 was seen and three of the antibodies recognised additional components at Mr ~ 310000 and ~ 330000. Crossreactive material of similar molecular mass was also seen in rabbit heart and brain, and in the skeletal muscle of other species.

Introducing undergraduate students to scientific reports

Abstract The question of how and when to introduce undergraduates to primary research articles is a perennial problem. We describe here a series of exercises undertaken with Level One students as introductory training towards the reading and presentation of scientific papers at Level Three and the writing up of final year research projects. In the first exercise, students consider the structure of a scientific report and read and evaluate a given research paper. Subsequently, students are asked to imagine themselves as scientific investigators interested in a specific problem. In tutor-led group discussion, they design an experiment to investigate the problem and then individually write a report based on provided data.

An Exercise To Teach Bioscience Students about Plagiarism.

Plagiarism is an issue of increasing concern to educators, yet students are not always clear about the boundaries between acceptable and unacceptable practice. An exercise to help bioscience students make this important distinction is described.

Enhancing understanding of recombinant DNA technology

Study of recombinant DNA technology in schools has in the past been confined to the theoretical explanations of cutting and splicing DNA and of using plasmids. A DNA Technology Kit has now been developed in the Department of Biochemistry at the University of Leicester. It has been successfully tested, end is available for hiring out to local schools and colleges for teachers and sixth-form students to gain first-hand practical experience of restriction enzyme digestion, agarose gel electrophoresis, and analysis and interpretation of laboratory data using a specially-prepared recombinant plasmid. This paper presents the theoretical background to the principles underlying recombinant DNA technology, followed by the practical procedures using the kit, and the problem-solving activities which can be developed at A level.

Derivation and partial analysis of two highly active myeloma cell transfectants

Vectors have been designed to optimise the expression of heterologous proteins in transfected mouse myeloma cells. The over-ridingly important DNA element contained in these constructs is the classical mouse immunoglobulin heavy chain enhancer. It is shown that even in the absence of a well-known promoter element, the enhancer can drive gene expression in stable cell transfectants and the main transcriptional start site utilized in such situations has been mapped to within the previously defined enhancer region. Using chicken lysozyme as a reporter function in these vectors, two transfected myeloma cell clones have been isolated which secrete this protein at levels 50-100-times as high as those usually obtained with the same vectors and it is shown that in molar terms this is at least as high as endogenous immunoglobulin produced by a related line. Analysis of these lines show that in one case only a single copy, and in the other two to three copies, of the apparently unrearranged vector have integrated at a single locus within the genome. Possible explanations for the high-level expression are discussed.