Pseudovitamin D-deficiency rickets is caused by mutations in the cytochrome P450 enzyme, 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-OHase). Patients with the disease exhibit growth retardation, rickets, and osteomalacia. Serum biochemistry is characterized by hypocalcemia, secondary hyperparathyroidism, and undetectable levels of 1alpha,25-dihydroxyvitamin D(3). We have inactivated the 1alpha-OHase gene in mice after homologous recombination in embryonic stem cells. Serum analysis of homozygous mutant animals confirmed that they were hypocalcemic, hypophosphatemic, hyperparathyroidic, and that they had undetectable 1alpha,25-dihydroxyvitamin D(3). Histological analysis of the bones from 3-week-old mutant animals confirmed the evidence of rickets. At the age of 8 weeks, femurs from 1alpha-OHase-ablated mice present a severe disorganization in the architecture of the growth plate and marked osteomalacia. These results show that we have successfully inactivated the 1alpha-OHase gene in mice and established a valid animal model of pseudovitamin D-deficiency rickets.
Pseudovitamin D-deficiency rickets (PDDR) is an autosomal disease characterized by hyperparathyroidism, rickets, and undetectable levels of 1,25-dihydroxyvitaminD3 (1,25(OH)2D3). Mice in which the 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) gene was inactivated presented the same clinical phenotype as patients with PDDR and were used to study renal expression of the epithelial Ca2+ channel (ECaC1), the calbindins, Na+/Ca2+ exchanger (NCX1), and Ca2+-ATPase (PMCA1b). Serum Ca2+ (1.20+/-0.05 mM) and mRNA/protein expression of ECaC1 (41+/-3%), calbindin-D28K (31+/-2%), calbindin-D9K (58+/-7%), NCX1 (10+/-2%), PMCA1b (96+/-4%) were decreased in 1alpha-OHase-/- mice compared with 1alpha-OHase+/- littermates. Feeding these mice a Ca2+-enriched diet normalized serum Ca2+ levels and expression of Ca2+ proteins except for calbindin-D9K expression. 1,25(OH)2D3 repletion resulted in increased expression of Ca2+ transport proteins and normalization of serum Ca2+ levels. Localization of Ca2+ transport proteins was clearly polarized in which ECaC1 was localized along the apical membrane, calbindin-D28K in the cytoplasm, and calbindin-D9K along the apical and basolateral membranes, resulting in a comprehensive mechanism facilitating renal transcellular Ca2+ transport. This study demonstrated that high dietary Ca2+ intake is an important regulator of the renal Ca2+ transport proteins in 1,25(OH)2D3-deficient status and thus contributes to the normalization of blood Ca2+ levels.
Among its penicillin-binding proteins (PBPs), Enterococcus faecium possesses a low-affinity PBP5, PBP5fm, which is the main target involved in -lactam resistance. A 7.7-kb EcoRI chromosomal fragment of E. faecium D63r containing the pbp5fm gene was cloned and sequenced. Two open reading frames (ORFs) were found. A 2,037-bp ORF encoded the deduced 73.8-kDa PBP5fm, the amino acid sequences of which were, respectively, 99.8, 78.5, and 62% homologous to those of the low-affinity plasmid-encoded PBP3r of Enterococcus hirae S185r and the chromosome-encoded PBP5 of E. hirae R40 and Enterococcus faecalis 56R. A second 597-bp ORF, designated psrfm, was found 2.3 kb upstream of pbp5fm. It appeared to be 285 bp shorter than and 74% homologous with the regulatory gene psr of E. hirae ATCC 9790. Different clinical isolates of E. faecium, for which a wide range of benzylpenicillin MICs were observed, showed that the increases in MICs were related to two mechanisms. For some strains of intermediate resistance (MICs of 16 to 64 g/ml), the increased level of resistance could be explained by the presence of larger quantities of PBP5fm which had an affinity for benzylpenicillin (second-order rate constant of protein acylation [k ؉2 /K] values of 17 to 25 M ؊1 s ؊1) that remained unchanged. For the two most highly resistant strains, EFM-1 (MIC, 90 g/ml) and H80721 (MIC, 512 g/ml), the resistance was related to different amino acid substitutions yielding very-low-affinity PBP5fm) which were synthesized in small quantities. More specifically, it appeared, with a three-dimensional model of the C-terminal domain of PBP5fm, that the substitutions of Met-485, located in the third position after the conserved SDN triad, by Thr in EFM-1 and by Ala in H80721 were the most likely cause of the decreasing affinity of PBP5fm observed in these strains.The genus Enterococcus, closely related to the genus Streptococcus, is involved in different clinical infections (37). Apart from their physiological properties, enterococci also differ from streptococci in that they generally are naturally 10-to 1,000-fold less susceptible to penicillins than streptococci (37). It was demonstrated that the natural low susceptibility of enterococci to penicillin is linked to the presence of at least one high-molecular-mass penicillin-binding protein (PBP) which has a low affinity for -lactams (1, 17, 50). Enterococcus faecium appears to be the enterococcal species most resistant to -lactam antibiotics, for which there are a wide range of benzylpenicillin MICs (0.5 to Ն 64 g/ml) for clinical isolates (22,30,50). Recently it became obvious that a new population of clinical E. faecium isolates for which the MICs of benzylpenicillin were very high (256 to 512 g/ml) had emerged in different countries (15,22,23,30).Among laboratory mutants and clinical isolates of E. faecium, two mechanisms have been shown to be involved in the high-level resistance to benzylpenicillin. These strains produced either an increased quantity of the essential low-affinity PBP5fm (15, 17) o...
Abstract. Estrogen deficiency results in a negative Ca 2ϩ balance and bone loss in postmenopausal women. In addition to bone, the intestine and kidney are potential sites for estrogen action and are involved in Ca 2ϩ handling and regulation. The epithelial Ca 2ϩ channel ECaC1 (or TRPV5) is the entry channel involved in active Ca 2ϩ transport. Ca 2ϩ entry is followed by cytosolic diffusion, facilitated by calbindin-D 28K and/or calbindin-D 9k , and active extrusion across the basolateral membrane by the Na ϩ /Ca 2ϩ -exchanger (NCX1) and plasma membrane Ca 2ϩ -ATPase (PMCA1b). In this transcellular Ca 2ϩ transport, ECaC1 probably represents the final regulatory target for hormonal control. The aim of this study was to determine whether 17-estradiol (17-E 2 ) is involved in Ca 2ϩ reabsorption via regulation of the expression of ECaC1. The ovariectomized rat model was used to investigate the regulation of ECaC1, at the mRNA and protein levels, by 17-E 2 replacement therapy. Using real-time quantitative PCR and immunohistochemical analyses, this study demonstrated that 17-E 2 treatment at pharmacologic doses increased renal mRNA levels of ECaC1, calbindin-D 28K , NCX1, and PMCA1b and increased the protein abundance of ECaC1. Furthermore, the involvement of 1,25-dihydroxyvitamin D 3 in the effects of 17-E 2 was examined in 25-hydroxyvitamin D 3 -1␣-hydroxylase-knockout mice. Renal mRNA expression of calbindin-D 9K , calbindin-D 28K , NCX1, and PMCA1b was not significantly altered after 17-E 2 treatment. In contrast, ECaC1 mRNA and protein levels were both significantly upregulated. Moreover, 17-E 2 treatment partially restored serum Ca 2ϩ levels, from 1.63 Ϯ 0.06 to 2.03 Ϯ 0.12 mM. In conclusion, this study suggests that 17-E 2 is positively involved in renal Ca 2ϩ reabsorption via the upregulation of ECaC1, an effect independent of 1,25-dihydroxyvitamin D 3 .
Keratinocytes express high levels of 25OHD 1alpha-hydroxylase (1OHase). The product of this enzyme, 1,25-dihydroxyvitamin D (1,25(OH)(2)D), promotes the differentiation of keratinocytes in vitro suggesting an important role for this enzyme in epidermal differentiation. To test whether 1OHase activity is essential for keratinocyte differentiation in vivo we examined the differentiation process in mice null for the expression of the 1alphaOHase gene (1alphaOHase(-/-)). Heterozygotes for the null allele were bred, and the progeny genotyped by PCR. The epidermis of the 1alphaOHase(-/-) animals and their wild-type littermates (1alphaOHase(+/+)) were examined by histology at the light and electron microscopic level, by immunocytochemistry for markers of differentiation, and by function examining the permeability barrier using transepidermal water loss (TEWL). No gross epidermal phenotype was observed; however, immunocytochemical assessment of the epidermis revealed a reduction in involucrin, filaggrin, and loricrin-markers of differentiation in the keratinocyte and critical for the formation of the cornified envelope. These observations were confirmed at the electron microscopic level, which showed a reduction in the F (containing filaggrin) and L (containing loricrin) granules and a reduced calcium gradient. The functional significance of these observations was tested using TEWL to evaluate the permeability barrier function of the epidermis. Although TEWL was normal in the basal state, following disruption of the barrier using tape stripping, the 1alphaOHase(-/-) animals displayed a markedly delayed recovery of normal barrier function. This delay was associated with a reduction in lamellar body secretion and a failure to reform the epidermal calcium gradient. Thus, the 25OHD 1OHase is essential for normal epidermal differentiation, most likely by producing the vitamin D metabolite, 1,25(OH)(2)D, responsible for inducing the proteins regulating calcium levels in the epidermis that are critical for the generation and maintenance of the barrier.
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