Award details

An instantimager for autoradiographic quantitation of protein, RNA and DNA in mammalian tissues and isotopic accumulation in cellular expression systems

Reference	JRI08153
Principal Investigator / Supervisor	Professor Hari Hundal
Co-Investigators / Co-Supervisors	Professor Michael Rennie, Dr Peter Taylor
Institution	University of Dundee
Department	College of Life Sciences
Funding type	Research
Value (£)	12,500
Status	Completed
Type	Research Grant
Start date	01/12/1996
End date	01/05/1997
Duration	5 months

Abstract

Funds are being requested to purchase a Packard InstantImager for the shared use by members of the department's membrane transport group for the purpose of autoradiographic quantification of protein, RNA and DNA in mammalian tissues as well as for the analysis of isotopic accumulation in cellular expression systems. There are five major research programmes in which the new equipment is required. The first relates to a study of the biochemical and physiological properties of sugar transporters in skeletal muscle and is primarily aimed at gaining some understanding of the cellular components involved in the insulin-induced translocation of GLUT4 and in addressing the molecular basis of impaired glucose utilisation during a variety of insulin resistant circumstances. Another major aspect of this work is to investigate the functional role of GLUT5. Its localisation within the plasma membrane of human skeletal muscle and its limited expression in other tissues signifies that it may be important in regulating hexose transport across the muscle membrane during normal and pathological circumstances. The second major area of research in which the InstantImager would be particularly valuable relates to studies of regulation and expression of Na/K-ATPase isoforms in skeletal muscle. We are specifically interested in gaining an insight into (i) the mechanism(s) underlying the insulin stimulation of the muscle Na/K-ATPase and (ii) identifying defects in the cellular processing of the different isoforms of the Na/K-ATPase in rat and human skeletal muscle during circumstances when insulin resistance may be a prominent feature. The third programme of work involves an investigation of the mechanism of thyroid hormone (T3) uptake in isolated liver membrane vesicles and an assessment of the effects of hypo and hyperthyroidism on hepatic T3 uptake. This project also involves examining the effects of altered thyroid status on the expression and function of glucose transporters and Na/K-ATPase isoforms in liver and muscle membranes and requires quantification of the immunoreactive signals observed on nitrocellulose membranes. A fourth project involves investigations into function and regulation of the cationic amino acid transporter and of NBAT/4F2 related amino acid transporters through expression of epitope- tagged mutant CAT and NBAT cDNAs in Xenopus oocytes. As part of our functional assay, we currently have to solubilise oocytes expressing proteins of interest to us to quantify amino acid uptake, however, it is expected that the InstantImager will enable us to directly quantify the amount of labelled amino acid accumulated without oocyte disruption and thus allowing us to retain them for additional studies. The fifth programme of research in which the new equipment will be particularly useful involves the identification and characterisation, at the molecular level, of the cardiac glutamate transporter which may play an important role in regulating cellular pH during myocardial infarction. One important part of this work currently involves the use of appropriate probes to known glutamate and neutral amino acid transporters to screen a pig heart library. Conventional methods of library screening are proving to be very slow but could be substantially accelerated using the InstantImager which allows data acquisition in real time.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	Joint Equipment Research Initiative 1996 (JE1) [1996]
Funding Scheme	X – not Funded via a specific Funding Scheme

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