Percepciones sobre el nivel individual

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ATP dependent endocytic vésiculation was measured at 0-12.5mM ATP in ghosts from 19 normal individuals recruited trom the laboratory. Endocytic vésiculation was measured by reduction in AChE activity using the 96 well plate method. Ghosts were made from controls and were incubated with 0-12.5mM ATP.

Table 4.1 Day-to-day (rows) and interindividual (columns) variations in ATP-dependent

endocytic vésiculation in normal controls.

ATP-dependent endocytic vésiculation: % reduction in AChE

Experiment (day): Jg37 Jg40 Jg41 Jg42 Jg45 Mean SD Normal control (Code name) A 21 26 22 23 2.16 B 35 31 33 2 C 34 22 18 25 6.8 D 31 28 24 29 28 2.55 E 33 29 31 24 26 29 3.26 F 47 35 41 6 G 35 35 0 H 24 24 0 I 28 28 0 J 29 25 27 2 K 23 23 0 L 24 24 0 M 24 24 0 N 21 21 0 O 19 19 0 P 25 25 0 Mean 32 29 28 23 24 27 SD 7.24 4.03 4.3 2.31 3.27 5.41

ATP-dependent endocytic vésiculation in normal control ghosts.

ATP dependent endocytic vésiculation was measured at 5mM ATP in ghosts from normal individuals recruited from the laboratory. Endocytic vésiculation was measured by reduction in AChE activity using the 96 well plate method. Ghosts were made from controls and were incubated with 5mM ATP.

Variations between different controls performed at the same time are shown in the 5 columns, jg37, 40, 41, 42 and 45, each representing a separate experiment (performed on separate days). Day-to-day variations from the same individual are shown in the rows A to P, each letter representing a different individual. Letter E represents the individual who was used as a control for the patient data most of the time. The mean value of control E (29%) is close to the mean value from all the individuals (27%) showing that this was a representative control.

4.4.2. Experiments with OHSt patients

The effect of DMA on normal cells was examined initially. Washed whole

erythrocytes from 8 normal controls recruited from the laboratory were incubated

with 2mM DMA at 37°C for 30 min before the preparation of phosphate-free

ghosts and the measurement of ATP-dependent endocytic vésiculation (Figure

4.4). The data illustrate that incubation of whole erythrocytes with 2mM DMA

had no effect on ATP-dependent endocytic vésiculation.

In the vast majority of patient studies, ATP-dependent endocytic

vésiculation was measured over the concentration range of 0-12.5mM ATP.

However, in the preliminary OHSt patient experiments, only 5mM ATP was used

(since this was the value at which maximal endocytic vésiculation occurred in

normal controls). Some initial experiments using DMA or DMP (dimethyl

pimelimidate) were performed at the same time. Table 4.2 presents these data. In

both the Manchester and Brighton pedigrees, which are typical OHSt pedigrees, it

appears that the patients had a complete defect in endocytosis. This defect was

corrected by DMA, but not by DMP. Endocytosis always occurred with control

erythrocyte ghosts, and was unaffected by DMA or DMP. These findings are

consistent with those of Mentzer et al. (1978). DMA and DMP differ solely in the

the same reactive groups at the end. Their structures are shown in Figure 1.7

(Chapter 1).

Since it was possible that these results might have occurred because the

OHSt cells had a different affinity for ATP, it was decided to look at a wide range

of ATP concentrations. Figure 4.5 illustrates absence of ATP-dependent

endocytic vésiculation in the Brighton pedigree using the range from 0-12.5mM

or 15mM ATP. These results confirm that ATP-dependent endocytic vésiculation

does not occur to any significant extent in the Brighton pedigree at any ATP

concentration up to 15mM.

Figure 4.6 shows the effect of incubating whole erythrocytes with varying

concentrations of DMA on (a) the ghost endocytosis and (b) the

ouabain4-bumetanide-resistant influx in control cells and those from a patient

(Manchester pedigree, patient B-III-2) with OHSt. The data show that DMA

corrected both the defect in endocytic vésiculation and the 'leak' influx almost

to a normal level. In the control, DMA had no effect on endocytic vésiculation

(also shown in Figure 4.4) and had no effect on the 'leak' influx (also shown

in table 4.3).

To determine whether DMA can act on the washed, broken membranes,

dependent endocytic vésiculation at 5mM ATP was measured. MOPS lysis buffer

was used to substitute for Tris lysis buffer at all stages. This was because the Tris

reacts with DMA and stops it from working. Figure 4.7 shows the effect of 0-1

mM DMA on the actual membranes in control ghosts and those from another

patient (Brighton) with OHSt. The DMA had no effect on ATP-dependent

endocytic vésiculation when incubated with the membranes in this experiment.

The Harrow case was haematologically similar to the two original OHSt

pedigrees, but not identical. The haemoglobin measurement was not as low as the

others were and the MCV was not so high. The deficiency of stomatin is less than

that of the original cases (Chetty, Stewart, unpublished). In this atypical OHSt

Harrow case, there was no obvious defect in ATP-dependent endocytic

vésiculation (Figure 4.8) though it was slightly lower than the parallel control.

DMA had no effect on endocytic vésiculation in the patient's cells (Figure 4.9)

and had no effect on the 'leak' influx (Table 4.3). This showed that endocytic

vésiculation can be present in OHSt cases and that if it is present, then 2mM

Figure 4.4 Effect of 2mM DMA on ATP-dependent endocytic vésiculation