Ernest G. Loten

Preparation of rat liver plasma membranes in a high yield

Existing procedures for the preparation of rat liver plasma membranes are time consuming and generally produce low yields. A method is described in which a rat liver homogenate low-speed pellet is fractionated on a self-forming Percoll gradient. Plasma membranes can be removed from the gradient in a high yield along with much of the DNA in the liver homogenate. A second Percoll step performed in the presence of a low concentration of calcium ions separates the DNA from the plasma membranes. The final membrane fraction has high specific activities of marker enzymes with little contamination with microsomal, mitochondrial, Golgi, or lysosomal markers.

Curriculum reform at the University of Otago Medical School.

The University of Otago Medical School, the older of the two medical schools in New Zealand, identified during the 1980s many of the same problems with its undergraduate curriculum as were reported in the United States, Canada, and the United Kingdom. An early, overly ambitious attempt to introduce a full problem-based learning curriculum at Otago failed; however, many piecemeal changes that embodied some of the principles of problem-based learning were successfully implemented. Subsequently, as desire for more coordinated and substantive change grew, Otagos faculty used what they had learned from their first effort to successfully introduce a modular systems-based preclinical curriculum in 1997. The authors describe the features of the new curriculum and discuss two components (a systems-integration course and a large-scale program of computerized in-course testing) that are particularly innovative. The new curriculum is already achieving one of its main goals (increasing the perceived relevance of preclinical teaching) and other outcomes are being evaluated.

Brief problem-solving questions in medical school examinations: is it necessary for students to explain their answers?

To compare outcomes when answers to objective type problem‐solving questions are marked with and without consideration of students’ explanations of their answers.

A rapid purification of hepatic ATP citrate lyase using blue sepharose

The lipogenic enzyme ATP citrate lyase (EC 4.1.3.8) has been shown to be phosphorylated in response to hormonal stimulation. Insulin and glucagon have been demonstrated to selectively stimulate phosphorylation of hepatocyte ATP citrate lyase [ 1,2] and insulin has a similar effect on adipocyte ATP citrate lyase [3]. Furthermore, ATP citrate lyase purified from rat mammary gland has been phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase [4]. The methods available for the purification of ATP citrate lyase from liver are either time consuming [5] or utilize hepatocytes as the starting material [ 11. Here, we present a rapid method for the purification of ATP citrate lyase starting with intact liver. The method is an extension of that in [l] in that the procedure for hepatocytes is adapted for use with whole liver by using blue Sepharose as an additional purification step. Benzamidine, a protease inhibitor, is used in order to minimize proteolytic degradation of the lyase during purification. The ATP citrate lyase prepared from whole liver using this method is recovered with a slightly higher specific activity and in a better yield than that obtained from hepatocytes [ 11. Furthermore, it is completely free from the endogenously generated proteolytic fragment (M, 57 000) that has been observed as a major contaminant [ 11.

The effect of phenoxybenzamine on the relationship between glycerol release and cyclic AMP levels in the presence of insulin in epinephrine-stimulated rat adipocytes

Insulin does not affect the relationship of cellular cyclic AMP levels to glycerol in rat adipocytes stimulated with corticotropin [l]. The inhibitory action of insulin on lipolysis was shown to fully account for the decrease in cyclic AMP. This was true, irrespective of whether measurements were made when cyclic AMP was on the upward rise after hormone stimulation, or on the decline. The curves obtained with and without insulin were superimposable. On the other hand, in the presence of ep~eph~ne, a mixed (Yand &agonist, the drop in glycerol production due to insulin, has been shown to be greater than expected from the observed fall in cyclic AMP levels [2]. Phenoxybenzamine, an a-adrenergic blocker, in the concentration range l-l 0 PM, has been shown to have a potentiating effect on epinephr~e-stimulated lipolysis in rat fat cells [3f. We investigate here whether this effect of phenoxybenzamine could modify the effects of epinephrine on lipolysis and cyclic AMP levels to alter their relationship sufficiently so that the decrease in lipolysis due to insulin can be fully accounted for by the observed fall in cyclic AMP.

Effect of remedial tutorial help on students who fail in-course assessments

No abstract available.

Activation and inhibition of insulin receptor autophosphorylation by trypsin treatment of intact H35 cells.

1. Treatment of intact cultured H35 cells with trypsin (1 mg/ml) for 15 min at low temperature (4 degrees C) or for 30 sec at 37 degrees C causes activation of the insulin receptor subsequently isolated from the cells. 2. Receptor activation was assessed by increased phosphotyrosine content of the beta-subunit of the receptor, and increased autophosphorylation using [32P]-ATP. 3. Treatment of the cells for 15 min at 37 degrees C however completely abolished insulin binding and all insulin receptor kinase activity. 4. These data demonstrate that proteolytic damage of the extracellular domain of the insulin receptor can render the receptor kinase inactive and lead to a cell which is unresponsive to insulin.

Detergent solubilisation of rat liver particulate cyclic AMP phosphodiesterase

Detergent solubilisation of particulate rat liver low Km cyclic AMP phosphodiesterase in the presence of protease inhibitors yields a form of the enzyme with a larger molecular weight than the form solubilised by protease treatment. The detergent solubilised enzyme could be partially purified by anion exchange chromatography. It displayed a marked tendency to precipitate from solution when detergent was removed.

Partial purification of protease-solubilised low Km cyclic nucleotide phosphodiesterase from liver

Abstract 1. 1. A particulate low Km phosphodiesterase from liver was solubilised by chymotrypsin treatment of liver particles and purified nearly 3000-fold. 2. 2. Whereas the crude or partially-purified enzyme showed curved Lineweaver-Burk kinetic plots, the purified enzyme showed Michaelis-Menten kinetics and had a Hill coefficient of 1.00.

Stimulation of low Km cyclic AMP phosphodiesterase by sulphydryl modification

Low concentrations of the organic mercurials, p-chloromercuribenzoate or p-chloromercuriphenylsulphonate activate the particulate low Km phosphodiesterase from adipose tissue and liver. Higher concentrations are inhibitory. Enzyme which has been activated by treatment of adipocytes with insulin, is not activated by the organic mercurials although inhibition by higher concentrations is seen. Enzyme from non-insulin treated adipocytes is activated and solubilised by mild trypsin treatment. Enzyme activated by either insulin treatment, or by p-chloromercuribenzoate is not further activated by trypsin, but it is solubilised.