PARA LAS HIPÓTESIS ESPECÍFICAS

There is considerable disagreement in the literature about the levels of

biosynthesis of LPH and the levels of LPH mRNA observed in rats. Early studies by

Jonas et a l (1985) showed the rate of biosynthesis of lactase relative to that of other microvillar membrane proteins to be reduced in adult rats. In contrast, other workers

repoited similar rates of lactase synthesis and insertion into the membrane in the new­

born and adult rat but suggested a change in processing to a catalytically inactive form of

apparent molecular weight 100,(KK) (Quan et a l, 1990; Castillo et a l , 1990). Another group detected a band of apparent size 110,(K)0 in adult rats in addition to the mature

LPH band and also observed a fucosylated fomi of apparent molecular weight 300,000

using a polyclonal anti-sera (Nsi-Emvo et a l , 1987). The fucosylated fonn was present at higher amounts on the intracellular membranes of adult than pre-weaned rats and these

lactase activity. However, Castillo et al. (1990) suggested that this larger component may be an aggregate of the protein of apparent molecular weight 100,000 since the

electi'ophoresis conditions used did not involve dénaturation. In all these studies on adult

rats the mature fonn of lactase was detected. Other workers have observed an accelerated

turn over rate of lactase protein using metabolic labelling studies in post weaned versus

preweaned rats (Tsuboi etaL, 1992).

If the decline of lactase activity was controlled at the level of transcription of the

LPH mRNA then the level of activity would be expected to parallel the amount of

message obseiwed. This did appear to be the case in a study of Sebastio et al. where the level of LPH mRNA, standardised using p-actin mRNA, was reported to be higher in

new-born than in adult rats (Sebastio et al., 1989). However, Freund et al. (1989) demonstrated the same level of lactase mRNA in adult and young rats by Northern blot

analysis, when villin mRNA was used as the control. This high level of LPH mRNA

throughout life would suggest post translational control is important in the decline of

lactase activity in adulthood.

Using a veiy different approach Buller et al. (1990) assessed lactase mRNA levels by Northern and dot blot analysis and presented the results as a proportion of total

RNA content of the intestine, since the levels of both p-actin and villin change during

development. These authors had previously obseiwed that the total lactase activity in the

intestine was actually higher in adult than in new-born rats, but the total protein in the

small intestine had increased dramatically such that the specific activity of lactase was

lower in adults than in new-borns (mU/mg protein) (Buller et al., 1989). Even though appreciable levels of lactase mRNA were found in all ages of rat, when measured relative

to total mRNA content the pattern of mRNA level minored that of total lactase activity,

which they took to imply transcriptional control (Buller et al., 1990).

More recently, Freund et al. (1991) have demonstrated heterogeneity of the expression of lactase in the intestine of rats. Using p actin mRNA as the control, they

showed post translational control of lactase in the jejunum and pre-translational regulation

in the colon (Freund et al., 1991). Indeed, it has been shown that lactase mRNA is not detectable in the distal ileum of adult rats (Freund etaL, 1991 and 1993). The restriction of the region in which LPH mRNA is detected in adult as compared to pre-weaned rats

means that the whole intestine approach taken by Buller et al. (1990) may not accurately portray the relationship between lactase protein and mRNA in the jejunum. Indeed, when

a similar study to that of Buller et al. (1990), who had concluded that transcriptional control was important, was peiformed using the jejunum only, the conclusion was that

translational control was important (Nudell et al., 1993). In this study a 7S RNA was used as the control and the results were reported to be similar when expressed in terms of

total RNA content of the jejunum. The level of LPH mRNA was found to increase

during development even when the level of LPH activity was decreasing. Lactase mRNA

reached a maximal proportion of the total enterocyte mRNA pool at day 22 (assessed

using 7S RNA as the internal developmental control), whereas the activity was maximal

in the neonate (Nudell et al., 1993).

In situ hybridisation to LPH-RNA in fetal rats initially demonstrated co­

localisation of the lactase protein and the mRNA (Rings et a i , 1992b). Analysis of a series of fetal ages showed that the lactase message was detectable 2 days before any

protein and that at birth the lactase mRNA was found only in the lower portion of the

villus whereas lactase protein was detected over the whole villus (Rings et al., 1992a). In adult rats, LPH-mRNA was only abundant in the middle section of the intestine (Rings

et al., 1993). Interestingly, these authors noted a short region of mosaic expression of lactase in the duodenum.

In rats, unlike rabbits all the evidence to date suggests that lactase is encoded by a

1.1.8.3.3. S tu d ie s u sin g h u m a n in te s tin e .

In humans there is conflicting evidence as to whether the mRNA level is high or

low in lactase non-persistent individuals. Sebastio et al. (1989) using slot-blot and SI protection assays demonstrated less LPH mRNA in non-persistent individuals than in

lactase persistent individuals, when p-actin was used to normalise for the amount of

RNA loaded. However, some non-persistent individuals in this study showed very high

mRNA levels and there was a poor correlation between activity and mRNA level in the

adult. In contrast, other groups have found that all individuals who were non-persistent

have low LPH mRNA levels as assessed by Northern blot analysis, in which actin

mRNA was examined as a control (Lloyd et a!., 1992; Escher et a i , 1992). Thus these studies, in contrast to that of Sebastio and colleagues, seemed to suggest that regulation at

the level of transcription of the lactase gene is impoitant in the lactase persistence / non­

persistence polymorphism.

It is noteworthy that the level of biosynthesis of LPH and the protein processing

in lactase non-persistent individuals appears to be heterogeneous. One of the four

individuals studied by Witte et al. showed an alteration in the rate of conversion of lactase precursor to the mature form, whereas the other three individuals demonstrated reduced

synthesis of the precursor protein (Witte et al., 1990). Sterchi et al. (1990) demonstrated lower levels of biosynthesis in all the non-persistent individuals in their study population

than in the persistent individuals. However, they observed slower maturation of this

protein and accumulation in the Golgi apparatus in some instances (Sterchi et a i, 1990). In a study using metabolic labelling of explants Rossi et al. (1993) demonstrated a considerable degree of heterogeneity in their Neapolitan study population. A group

(6/32) of the non-persistent individuals were observed to incorporate as much label as the

persistent individuals, however it is not known whether this was due to high rate of

Analysis of the expression of lactase at a cellular level in humans has been

peiformed using techniques to detect the enzyme activity, the protein and the mRNA.

Immunoreactive LPH protein was detected in all the lactase persistent individuals

examined. However, two classes of lactase non-persistent individuals were found, some

showing no brush border staining for lactase, but continuous brush border staining for

sucrase-isomaltase (SI) while others exhibited a mosaic pattern of staining of the brush

border with all the anti-lactase monoclonal antibodies tested but continuous staining for

SI (Maiuri et aL, 1991). In a further study enzymohistochemistiy and

immunohistochemistry were peifonned on sections from the same individuals and it was

shown that the activity and protein were coincident (Maiuri et a i , 1993a). Using intact pieces of intestine it was shown that in lactase non-persistent adults patches of lactase

positive enterocytes are scattered on the villus surface (Maiuri et aL, 1993a). It was not possible to explain this phenomenon by the clonal origin of the enterocyte populations

emerging from the ciypts since ribbons of positively staining cells would have been

expected. This is in contrast to the situation in adult rabbits where ribbons of positively

staining cells were seen (section 1.1.8.3.1)(Maiuri et aL, 1993a). It is also of interest that these investigators have shown patchy expression of the blood group A antigen in the

intestine of non-secretor individuals who were blood group A, which again could not be

attributed to the clonal origin of the enterocytes (Maiuri et aL, 1993b).

Maiuri et al. (1994) have perfomied in situ hybridisation on persistent and non- persistent individuals. All persistent individuals were found to express lactase mRNA in

most cells, with the peak in lactase mRNA quantity being lower down the villus than the

activity peak. In lactase non-persistent individuals a variety of different cell types were

found, namely cells that express the mRNA, protein and activity, cells that have mRNA

and protein but no activity and cells which possess the mRNA but do not synthesis the

oo ATG Alu elements TATA box Start of mature

lactase protein Poly A site

I

i

f-h*

10 kilobases

Figure 1.1.9.1.