Ontogeny of intestinal safety factors: lactase capacities and lactose loads.
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We measured intestinal safety factors (ratio of a physiological capacity to the load on it) for lactose digestion in developing rat pups. Specifically, we assessed the quantitative relationships between lactose load and the series capacities of lactase and the Na+-glucose cotransporter (SGLT-1). Both capacities increased significantly with age in suckling pups as a result of increasing intestinal mass and maintenance of mass-specific activities. The youngest pups examined (5 days) had surprisingly high safety factors of 8-13 for both lactase and SGLT-1, possibly because milk contains lactase substrates other than lactose; it also, however, suggests that their intestinal capacities were being prepared to meet future demands rather than just current ones. By day 10 (and also at day 15), increased lactose loads resulted in lower safety factors of 4-6, values more typical of adult intestines. The safety factor of SGLT-1 in day 30 (weanling) and day 100 (adult) rats was only approximately 1.0. This was initially unexpected, because most adult intestines maintain a modest reserve capacity beyond nutrient load values, but postweaning rats appear to use hindgut fermentation, assessed by gut morphology and hydrogen production assays, as a built-in reserve capacity. The series capacities of lactase and SGLT-1 varied in concert with each other over ontogeny and as lactose load was manipulated by experimental variation in litter size. (+info)
Species differences in the sites of cleavage of pro-lactase to lactase supports lack of selective pressure.
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The pro-sequences in pro-lactase-phlorizin hydrolase (LPH) are needed for lactase to proceed past the ER, but are irrelevant as to the enzymatic activities. Hence, in all species removal of the pro- sequences (or most of them) must take place after the ER. Contrary to this, the details of the removal of these pro-sequences are to be expected to differ in the various species, since they are not subjected to selective pressure. Using site-directed mutagenesis we investigated processing in rabbit. The first cleavage occurs by furin (or furin-like PCs) and takes place at R-A-A-R(349) in the pro-sequence, generating the known 180 kDa intermediate. Replacing R(349) by Q results in a mutant which is not cleaved but nevertheless transported to the cell surface as demonstrated by immunofluorescence. Further processing of either the 180 kDa intermediate or the mutant is not directly mediated by furin-like PCs, but involves (also) other proteases. These results demonstrate that formation of the 180 kDa intermediate, consistently found only in rabbits, but not in man, is not essential for lactase transport: in all likelihood lack of selective pressure has led to species-specific processing of pro-LPH. (+info)
Loss of Hoxa5 gene function in mice perturbs intestinal maturation.
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The Hox gene family of transcription factors constitutes candidate regulators in the molecular cascade of events that governs establishment of normal terminal differentiation along the duodenum to colon axis. One member of this family, Hoxa5, displays a dynamic pattern of expression during gut development. Hoxa5 transcripts are present in midgut mesenchyme at the time of remodeling, supporting a role for this gene in digestive tract specification. To study the role of Hoxa5 in proper intestinal development and maturation, we examined whether Hoxa5 mutant mice exhibit any defect in this process. We report here that even though Hoxa5 is not required for midgut morphogenesis, its loss of function perturbs the acquisition of adult mode of digestion, which normally is temporally coordinated with the process of spontaneous weaning. Impaired maturation of the digestive tract might be related to altered specification of intestinal epithelial cells. Our findings provide evidence that Hoxa5 expression in the gut mesoderm is important for the region-specific differentiation of the adjacent endoderm. (+info)
Common polymorphism in a highly variable region upstream of the human lactase gene affects DNA-protein interactions.
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In most mammals lactase activity declines after weaning when lactose is no longer part of the diet, but in many humans lactase activity persists into adult life. The difference responsible for this phenotypic polymorphism has been shown to be cis-acting to the lactase gene. The causal sequence difference has not been found so far, but a number of polymorphic sites have been found within and near to the lactase gene. We have shown previously that in Europeans there are two polymorphic sites in a small region between 974 bp and 852 bp upstream from the start of transcription, which are detectable by denaturing gradient gel electrophoresis (DGGE). In this study, analysis of individuals from five other population groups by the same DGGE method reveals four new alleles resulting from three additional nucleotide changes within this very small region. Analysis of sequence in four primate species and comparison with the published pig sequence shows that the overall sequence of this highly variable human region is conserved in pigs as well as primates, and that it lies within a 1kb region which has been shown to control lactase downregulation in pigs. Electrophoretic mobility shift assay (EMSA) studies were carried out to determine whether common variation affected protein-DNA binding and several binding activities were found using this technique. A novel two base-pair deletion that is common in most populations tested, but is not present in Europeans, caused no change in binding activity. However, a previously published C to T transition at -958bp dramatically reduced binding activity, although the functional significance of this is not clear. (+info)
Combined effects of trehalose and cations on the thermal resistance of beta-galactosidase in freeze-dried systems.
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The purpose of this study was to investigate the combined effects of trehalose and cations on the preservation of beta-galactosidase in freeze-dried systems and their relationship to physical properties. Differential scanning calorimetry was employed to measure the glass transition temperature (T(g)) and the endothermal peak area, related to the amount of crystalline trehalose dihydrate present in the samples. In systems in which the trehalose matrix was humidified to conditions which allowed a high proportion of trehalose to crystallize, the enzyme was rapidly inactivated upon heating at 70 degrees C. In these conditions the addition of CsCl, NaCl and particularly KCl or MgCl(2), improved the enzyme stability with respect to that observed in matrices containing only trehalose. For a given moisture content, addition of salts produced very little change on the glass transition temperature; therefore the protective effect could not be attributed to a higher T(g) value. The crystallization of trehalose dihydrate in the humidified samples was delayed in the trehalose/salt systems (principally in the presence of Mg(2+)) and a parallel improvement of enzyme stability was observed. (+info)
Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase.
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Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane-bound, family 1 beta-glycosidase found on the brush border of the mammalian small intestine. LPH, purified from sheep small intestine, was capable of hydrolysing a range of flavonol and isoflavone glycosides. The catalytic efficiency (k(cat)/K(m)) for the hydrolysis of quercetin-4'-glucoside, quercetin-3-glucoside, genistein-7-glucoside and daidzein-7-glucoside was 170, 137, 77 and 14 (mM(-1) s(-1)) respectively. The majority of the activity occurred at the lactase and not phlorizin hydrolase site. The ability of LPH to deglycosylate dietary (iso)flavonoid glycosides suggests a possible role for this enzyme in the metabolism of these biologically active compounds. (+info)
Mitochondrial ATP production is necessary for activation of the extracellular-signal-regulated kinases during ischaemia/reperfusion in rat myocyte-derived H9c2 cells.
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To search for the stimuli involved in activating the mitogen-activated protein kinases (MAPKs) during ischaemia and reperfusion, we simulated the event in a system in vitro conducive to continuous and non-invasive measurements of several major perturbations that occur at the time: O(2) tension, mitochondrial respiration and energy status. Using H9c2 cells (a clonal line derived from rat heart), we found that activation of the extracellular signal-regulated MAPKs (ERKs) on reoxygenation was abolished if the mitochondria were inhibited prior to and during reoxygenation. Re-introduction of O(2) per se is therefore not sufficient to activate the ERKs. Recovery and maintenance of cellular ATP levels by mitochondrial respiration is necessary, although ATP recovery alone is not sufficient. ERK activation by H(2)O(2), but not phorbol esters, was also sensitive to mitochondrial inhibition. Thus, reoxygenation and H(2)O(2)-mediated oxidative stress share a mechanism of ERK activation that is ATP- or mitochondrion-dependent, and this common feature suggests that the reoxygenation response is mediated by reactive oxygen species. A correlation between ERK activity and ATP levels was also found during the anoxic phase of ischaemia, an effect that was not due to substrate limitation for the kinases. Our results reveal the importance of cellular metabolism in ERK activation, and introduce ATP as a novel participant in the mechanisms underlying the ERK cascade. (+info)
Lactase haplotype diversity in the Old World.
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Lactase persistence, the genetic trait in which intestinal lactase activity persists at childhood levels into adulthood, varies in frequency in different human populations, being most frequent in northern Europeans and certain African and Arabian nomadic tribes, who have a history of drinking fresh milk. Selection is likely to have played an important role in establishing these different frequencies since the development of agricultural pastoralism approximately 9,000 years ago. We have previously shown that the element responsible for the lactase persistence/nonpersistence polymorphism in humans is cis-acting to the lactase gene and that lactase persistence is associated, in Europeans, with the most common 70-kb lactase haplotype, A. We report here a study of the 11-site haplotype in 1,338 chromosomes from 11 populations that differ in lactase persistence frequency. Our data show that haplotype diversity was generated both by point mutations and recombinations. The four globally common haplotypes (A, B, C, and U) are not closely related and have different distributions; the A haplotype is at high frequencies only in northern Europeans, where lactase persistence is common; and the U haplotype is virtually absent from Indo-European populations. Much more diversity is seen in sub-Saharan Africans than in non-Africans, consistent with an "Out of Africa" model for peopling of the Old World. Analysis of recent recombinant haplotypes by allele-specific PCR, along with deduction of the root haplotype from chimpanzee sequence, allowed construction of a haplotype network that assisted in evaluation of the relative roles of drift and selection in establishing the haplotype frequencies in the different populations. We suggest that genetic drift was important in shaping the general pattern of non-African haplotype diversity, with recent directional selection in northern Europeans for the haplotype associated with lactase persistence. (+info)