25 Hydroxyvitamin D 1 alpha-hydroxylase is required for optimal epidermal differentiation and permeability barrier homeostasis.

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.

Targeted inactivation of the 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets.

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.

Correction of the abnormal mineral ion homeostasis with a high-calcium, high-phosphorus, high-lactose diet rescues the PDDR phenotype of mice deficient for the 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1).

Mutations in the 25-hydroxyvitamin D-1alpha-hydroxylase gene (CYP27B1; 1alpha-OHase) cause pseudo vitamin D deficiency rickets (PDDR), while mutations in the vitamin D receptor (VDR) cause hereditary vitamin D resistance rickets. Animal models of both diseases have been engineered. The bone phenotype of VDR-ablated mice can be completely rescued by feeding the animals with a high-calcium, high-phosphorus, high-lactose diet. We have attempted to rescue the PDDR phenotype of mice deficient for the 1alpha-OHase gene by feeding them with the high-calcium diet. The rescue regimen consisted of feeding a diet containing 2% calcium, 1.25% phosphorus, 20% lactose (rescue diet) from 3 weeks of age until sacrifice at 8.5 weeks of age. Blood biochemistry analysis revealed that the rescue diet corrected the hypocalcemia and secondary hyperparathyroidism. Despite the restoration of normocalcemia, 1alpha-OHase(-/-) (and 1alpha-OHase(+/-)) animals fed the rescue diet initially gained weight less rapidly than control mice fed normal mouse chow. Although 1alpha-OHase(-/-) mice fed the rescue diet eventually reached the same weight as control animals, the treatment did not entirely correct bone growth, as femur size remained significantly smaller than that of control. Bone histology and histomorphometry confirmed that the rickets and osteomalacia were cured. The rescue diet also restored the biomechanical properties of the bone tissue within normal parameters. These results demonstrate that correction of the abnormal mineral ion homeostasis by feeding with a high-calcium rescue diet is effective to rescue the PDDR phenotype of 1alpha-OHase mutant mice. This treatment, however, does not appear as effective as 1,25(OH)(2)D(3) replacement therapy since bone growth remained impaired.

Mice lacking 25OHD 1alpha-hydroxylase demonstrate decreased epidermal differentiation and barrier function.

Keratinocytes express high levels of 25OHD 1alpha-hydroxylase (1OHase). The product of this enzyme, 1,25(OH)(2)D, promotes the differentiation of keratinocytes in vitro. 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(-/-)) by light and electron microscopy, by immunocytochemistry for markers of differentiation, by ion capture cytochemistry for calcium localization, and by function using transepidermal water loss (TEWL) to assess barrier integrity. Levels of involucrin, filaggrin, and loricrin-markers of differentiation in the keratinocyte and critical for the formation of the cornified envelope-were reduced in the epidermis of 1alphaOHase(-/-) mice. Calcium in the outer epidermis was reduced with loss of the calcium gradient from stratum basale to stratum granulosum. TEWL was normal in the resting state, but following disruption of the barrier, 1alphaOHase(-/-) mice had a markedly prolonged recovery of barrier function associated with a reduction in lamellar body secretion and a failure to reform the calcium gradient. Thus 1,25(OH)(2)D is essential for normal epidermal differentiation, most likely by inducing the proteins and mediating the calcium signaling in the epidermis required for the generation and maintenance of the barrier.

The PBP 5 synthesis repressor (psr) gene of Enterococcus hirae ATCC 9790 is substantially longer than previously reported.

A reexamination of the nucleotide sequence of the psr gene of Enterococcus hirae revealed the presence of two additional nucleotides at residues 1190 and 1191. As a result, instead of a stop codon after 148 aa, the psr gene product would contain 293 aa residues. The revised size of the gene product was confirmed by subsequently cloning and expressing the psr gene in Escherichia coli. The derived amino acid sequence of the revised psr gene product was found to be similar to several other proteins in the combined GenBank/EMBL database. The protein products of some of these genes are thought to play regulatory role(s) in exo or capsular polysaccharide synthesis and/or in cell wall metabolism. All the putative homologs of the revised Psr appear to have a putative membrane-anchoring domain at their N-termini. Amino acid blocks with high degrees of similarity have been identified in the aligned sequences, and it is suggested that these common motifs could be of structural or functional importance.

The gene encoding the low-affinity penicillin-binding protein 3r in Enterococcus hirae S185R is borne on a plasmid carrying other antibiotic resistance determinants.

Two plasmid-derived NcoI DNA fragments of 14 and 4.5 kb, respectively, have been isolated from the multidrug-resistant strain Enterococcus hirae S185R and analyzed. The 14-kb fragment contains two inverted (L and R) IS1216 insertion modules of the ISS1 family. These modules define a Tn5466 transposon-like structure that contains one copy of the methylase-encoding ermAM conferring erythromycin resistance and one copy of the adenylyl-transferase-encoding aadE conferring streptomycin resistance. Immediately on the left side of IS1216L there occurs a copy of pbp3r encoding the low-affinity penicillin-binding protein (PBP) PBP3r, itself preceded by a psr-like gene (psr3r) that controls the synthesis of PBP3r. ermAM, aadE, and the transposase gene (tnp) of IS1216R have the same polarities, and these are opposite those of psr3r, pbp3r, and the tnp gene of IS1216L. The 4.5-kb fragment is a copy of the 4.5-kb sequence at the 5' end of the 14-kb fragment, although it is not a restriction product of the 14-kb fragment. It contains three genes with the same polarity: psr3r, pbp3r, and tnp in an IS1216 element. Because of the very high degree of identity (99%) with the chromosomal psrfm and pbp5fm genes of Enterococcus faecium D63R, it is proposed that both the psr3r and pbp3r genes were transferred from an E.faecium strain and inserted in a plasmid of E. hirae. E. hirae is the first known bacterial species in which a low-affinity PBP-encoding gene has been found to be plasmid borne.

Structure of the low-affinity penicillin-binding protein 5 PBP5fm in wild-type and highly penicillin-resistant strains of Enterococcus faecium.

Among its penicillin-binding proteins (PBPs), Enterococcus faecium possesses a low-affinity PBP5, PBP5fm, which is the main target involved in beta-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 micrograms/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 micrograms/ml) and H80721 (MIC, 512 micrograms/ml), the resistance was related to different amino acid substitutions yielding very-low-affinity PBP5fm variants (k+2/K < or = 1.5 M(-1) s(-1)) 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.