A simple spectrophotometric assay was developed for peroxisomal fatty acyl-CoA oxidase activity. The assay, based on the H2O2-dependent oxidation of leuco-dichlorofluorescein catalysed by exogenous peroxidase, is more sensitive than methods previously described. By using mouse liver samples, cofactor requirements were assessed and a linear relationship was demonstrated between dye oxidation and enzyme concentration. By using this assay on subcellular fractions, palmitoyl-CoA oxidase activity was localized for the first time in microperoxisomes of rat intestine. The assay was also adapted to measure D-amino acid oxidase activity, demonstrating the versatility of this method for measuring activity of other H2O2-producing oxidases.

The problem based learning (PBL) curriculum at Manchester emphasizes small-group work. This is supported through group assessment where students assess key aspects of their group's function. In the study described here the authors evaluated students' perceptions of both PBL group work and what a group assessment needs to assess. They aimed to produce a description of the cognitive and motivational influences on group process and unpack the ways they contribute to a successful PBL group so that the kinds of skills an effective assessment should assess could be identified. Focus groups and a questionnaire were used to generate the data. The focus group results indicate that students support PBL group work as a method of learning, and that those groups that work cooperatively are perceived as facilitating the most motivating learning environment. The students supported the assessment being summative and felt that it could be simplified to measure: behavioural skills contributing to maximizing motivation of the group process; and cognitive skills relating to the content of the group discussion. The questionnaire results also supported the use of a summative assessment of small-group work that evaluates the domains of group process and the content of the group discussion.

Sheets of mucosal epithelial cells were released from guinea pig small intestine after incubation with ethylenediaminetetraacetate . Cells in sheets retained their columnar shape for 24 hr at room temperature, and exclusion of nigrosine suggested they had intact plasma membranes . When sheets were disaggregated individual cells had normal morphology for at least 4 hr. During isolation 16% of the total protein and 24% of the total lactic dehydrogenase were lost from the cells, but subsequent enzyme leakage was low . Leakage increased with shaking, incubation at 37°C, or increasing the oxygen tension of the suspending medium, but was minimal when the Na+ : K+ ratio in the medium was 8 :1 and the osmolarity was high . Losses of particulate enzyme activities were negligible . Respiration was constant for up to 4 hr and was insensitive to calcium, bicarbonate, oxygen tension, and pH . It was inhibited by cyanide and iodoacetate and varied with the Na+ : K+ ratio of the extracellular fluid and the structural integrity of the cells . All preparations concentrated potassium and excluded sodium, but lost this ability if ouabain was added or cells were broken . Potassium-42 uptake was also sensitive to temperature, ouabain, and structural integrity. The preparations are being used to study cell metabolism in the intestinal epithelium.

During pregnancy and lactation in the rat the small intestine in general and the mucosal epithelium in particular gain weight. The specific activities of sucrase, lactate dehydrogenase and succinate-tetrazolium reductase remain constant and those of glucose 6-phosphate dehydrogenase and isocitrate dehydrogenase increase. There is no evidence that the reported decrease in absorption per unit area or weight of mucosal epithelium during pregnancy and lactation is due to decreases in enzyme activities within the epithelium. The pattern of enzyme change shows that the response of the gut to the stimuli of pregnancy and lactation must be a complex one, possibly involving increases in the specific activities of some enzymes.

BIOCHEMICAL SOCIETY TRANSACTIONS electron microscopical observations [ 3 I . for biochemical analysis of enzyme activity and some liver sections were stained with gallocyanin stain for image analysis using the Quantimet QU20 to measure the nuclear area and number of hepatocytcs (S. C. Price &L R. S. Ahmed. unpublished work).DEHP is known to cause hepatomegaly and induce both peroxisomal enzymes and a specific form of cytochrome 1'-450 [ 21. In these experiments, biochemical assays showed an increase in liver size, in peroxisomal enzymes ( Fig. 1 ) and, to a lesser extent, in total cytochrome 1'-450 (not illustrated) during first feeding and on subsequent treatment with the DEHP-containing diet. After reversal, liver size and peroxisomal enzymes decreased, but not to the control values.Electron microscopy clearly showed peroxisome proliferation and, t o a lesser extent, proliferation in mitochondria and smooth endoplasmic reticulum and reduction of rough endoplasmic reticulum in the livers of rats receiving the DEHP-containing diet at the time of death. In groups of animals treated with DEHP and then returned to control diet there remained some difference in numbers of peroxisomes and mitochondria between test and control groups. A more marked difference. however. was that in animals prcviously treated with DEHP the rough endoplasmic reticulum was reorganized into very tight stacks with reduced cisternal space.Image analysis of nuclear number and size showed the expected changes in cell size and ploidy with increasing age 131 and also that the increase in liver size was due to an increase in cell number, not in cell size. This is consistent with earlier results showing that treatment with DEHP leads first to hyperplasia, then to hypertrophy 141. The mean nuclear area was increased in animals actually receiving DEHP (Fig. 1 ) and examination o f the frequency distribution showed that this was due to an increase in tetraploid nuclei. On withdrawal of DEHP, both nuclear number and nuclear size returned towards control values.The results obtained in these experiments indicate first that if rats are treated with DEHP, then returned t o control diet, and then re-fed DEHP. liver size increases more rapidly than in rats never exposed to the compound. Secondly, the increase in cell number on treatment with DEHP is accompanied by an increase in ploidy. Thirdly, o n reversal, the reduction in liver weight is due to a fall in cell number and accompanied by a reduction in ploidy. While reductions in cell number have been observed in other tissues, such as the thyroid gland following withdrawal of chemical stimuli 151, it is generally thought that an increase in ploidy is irreversible so that the reduction in ploidy can only bc explained by selective loss of tetraploid cells or by a replacement of part of the population of liver cells by a new population.