Safety assessment of the post-harvest treatment of button mushrooms (Agaricus bisporus) using ultraviolet light.

Wild mushrooms are an excellent source of vitamin D. The presence of vitamin D in mushrooms is attributed to sunlight exposure, which catalyzes the conversion of fungal ergosterol to vitamin D2 via a series of photochemical/thermal reactions. Mushroom growers now incorporate UV light treatments during processing to produce mushrooms with levels of vitamin D that compare to those in wild mushrooms. Presented herein is a comprehensive review of information relevant to the safety of introducing vitamin D mushrooms, produced using UV light technologies, to the food supply. Historical reference to the use of UV light for production of vitamin D is discussed, and studies evaluating the nutritional value and safety of vitamin D mushrooms are reviewed. Traditional safety evaluation practices for food additives are not applicable to whole foods; therefore, the application of substantial equivalence and history-of-safe-use is presented. It was demonstrated that vitamin D in mushrooms, produced using UV light technologies, are equivalent to vitamin D in mushrooms exposed to sunlight, and that UV light has a long-history of safe use for production of vitamin D in food. Vitamin D mushrooms produced using UV light technologies were therefore considered safe and suitable for introduction to the marketplace.

Dietary 135-fold cholecalciferol supplementation severely disturbs the endochondral ossification in growing dogs.

The effects of excessive non-toxic dietary Vitamin D(3) supplementation on Ca homeostasis with specific effects on endochondral ossification and skeletal remodeling were investigated in a group of growing Great Dane dogs supplemented with cholecalciferol (Vitamin D(3); HVitD) versus a control group (CVitD) (1350 microg versus 11.4 microg Vitamin D(3) per kilogram diet) from 6 to 21 weeks of age. There were no differences between groups in plasma concentrations of total Ca, inorganic phosphate, growth hormone, and insulin-like growth factor I and no signs of Vitamin D(3) intoxication in HVitD. For the duration of the study in HVitD compared to CVitD, plasma levels of parathyroid hormone (PTH) decreased, calcitonin (CT) increased, 25-hydroxycholecalciferol [25(OH)D(3)] increased 30- to 75-fold, 24,25-dihydroxycholecalciferol [24,25(OH)(2)D(3)] increased 12- to 16-fold, and 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)] decreased by approximately 40%. The latter was attributed to the two-fold increased metabolic clearance rate in the HVitD versus CVitD accompanied by the absence of the anabolic effect of PTH on the production of 1,25(OH)(2)D(3). Fractional Ca absorption (alpha) did not differ between groups at 8 and 14 weeks of age, whereas at 20 weeks of age alpha increased by only 16.4% in HVitD compared to CVitD. Excessive non-toxic Vitamin D(3) supplementation resulted in decreased bone remodeling and focal enlargement of the growth plate with morphology resembling those induced by administration of CT. Hypercalcitoninemia and the imbalanced relationship between 1,25(OH)(2)D(3) and 24,25(OH)(2)D(3) are potent candidates for the disturbed endochondral ossification.

Moderate cholecalciferol supplementation depresses intestinal calcium absorption in growing dogs.

Hormonal regulation of calcium (Ca) absorption was investigated in a cholecalciferol (vitamin D(3))-supplemented group (hVitD) vs. a control group (cVitD) of growing Great Danes (100 vs. 12.5 micro g vitamin D(3)/kg diet). Although Ca intakes did not differ, fractional Ca absorption was significantly lower in the hVitD group than in the cVitD group. There were no differences in plasma concentrations of Ca, inorganic phosphate, parathyroid hormone, growth hormone or insulin-like growth factor I between groups. Plasma 25-hydroxycholecalciferol [25(OH)D(3)] concentrations were maintained in the hVitD dogs at the same levels as in the cVitD dogs due to increased turnover of 25(OH)D(3) into 24,25-dihydroxycholecalciferol [24,25(OH)(2)D(3)] and 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)]. In hVitD dogs, the greater plasma 24,25(OH)(2)D(3) concentration and the enhanced metabolic clearance rate (MCR) of 1,25(OH)(2)D(3) indicated upregulated 24-hydroxylase activity. The increased MCR of 1,25(OH)(2)D(3) decreased plasma 1,25(OH)(2)D(3) concentrations. In hVitD dogs, the greater production rate of 1,25(OH)(2)D(3) was consistent with the 12.9-fold greater renal 1alpha-hydroxylase gene expression compared with cVitD dogs and compensated to a certain extent for the accelerated MCR of 1,25(OH)(2)D(3). The moderately decreased plasma 1,25(OH)(2)D(3) concentration can only partially explain the decreased Ca absorption in the hVitD dogs. Intestinal vitamin D receptor concentrations did not differ between groups and did not account for the decreased Ca absorption. We suggest that 24,25(OH)(2)D(3) may downregulate Ca absorption.

Regulation of the procine 1,25-dihydroxyvitamin D3-24-hydroxylase (CYP24) by 1,25-dihydroxyvitamin D3 and parathyroid hormone in AOK-B50 cells.

The 24-hydroxylase is the enzyme responsible for the first step in the catabolism of 1,25-dihydroxyvitamin D3, the active form of vitamin D. This enzyme was shown to be upregulated by 1,25-dihydroxyvitamin D3 itself and downregulated by parathyroid hormone (PTH). Upregulation of 24-hydroxylase by 1,25-dihydroxyvitamin D3 has been characterized; however, the mechanism by which PTH acts to downregulate 24-hydroxylase expression remains unknown. Here we report the cloning of the porcine 24-hydroxylase, and show that 1,25-dihydroxyvitamin D3-stimulated 24-hydroxylase mRNA and activity are repressed by PTH in AOK-B50 cells, a porcine kidney proximal tubule cell line with stably transfected opossum PTH receptors. Forskolin mimicked the effects of PTH consistent with in vivo data, and suppression by PTH was not due to changes in VDR levels. The first 1400 bp of the 24-hydroxylase promoter were not able to mediate the effects of PTH on a reporter gene. In view of the above findings we concluded that AOK-B50 cells are a suitable model for further studying the mechanism of action of PTH on 24-hydroxylase mRNA.

Cloning of the mouse 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP1alpha) gene.

A genomic clone for 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase) was isolated from a mouse embryonic stem cell P1 genomic library. It contains nine exons spanning 4.8 kb from the transcriptional start site. All the intron insertion sites are identical to that of the human 1alpha-hydroxylase and human vitamin D(3) 25-hydroxylase genes. A polyadenylation signal AUUAAA that differs from the consensus sequence at the second residue was identified 16 bp downstream of the 3' end of the previously reported mouse cDNA. This element is the only common natural variant described. The 3' end of the gene was determined using a RACE technique. Three poly(A) addition sites were observed 12, 15 and 22 bases from the AUUAAA element. Such distances are in agreement with what is required for maturation of mammalian pre-mRNAs. This molecular cloning makes possible the generation of transgenic mice in order to further investigate the role and importance of the 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP1alpha).

Dietary calcium is a major factor in 1,25-dihydroxycholecalciferol suppression of experimental autoimmune encephalomyelitis in mice.

The active form of vitamin D (1,25-dihydroxycholecalciferol) is a potent immune system regulator. Treating mice with 1, 25-dihydroxycholecalciferol and feeding them diets high in calcium can completely suppress the induction of experimental autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE). Experiments described here were carried out on mice in which development of EAE was induced. Mice were fed diets containing various amounts of calcium and 1,25-dihydroxychole-calciferol. Variables measured were as follows: 1) incidence and severity of EAE; 2) serum calcium concentrations; 3) body weight; 4) total number of cells in the lymph nodes; and 5) interleukin-4 (IL-4) and transforming growth factor-beta1 (TGF-beta1) mRNA levels. When calcium was removed from the diet, the incidence of EAE was reduced 20% in both males and females. Further, the lower the dietary level of calcium, the higher was the dose of 1,25-dihydroxycholecalciferol required to prevent the symptoms. Thus, 1, 25-dihydroxycholecalciferol was found most effective in mice fed a diet adequate or high in calcium. 1,25-Dihydroxycholecalciferol treatment of mice fed high dietary calcium resulted in a decreased number of lymphocytes in the lymph nodes and increased IL-4 and TGF-beta1 mRNA levels. When calcium was omitted from the diet, 1, 25-dihydroxycholecalciferol supplementation increased TGF-beta1 mRNA. Increased IL-4 mRNA and decreased lymphocytes in the lymph nodes in response to 1,25-dihydroxycholecalciferol occurred only when dietary calcium was adequate or high. Our results suggest that dietary calcium and 1,25-dihydroxycholecalciferol are both involved in the prevention of symptomatic EAE.

A characterization of vitamin D-independent intestinal calcium absorption in the osteopetrotic (op/op) mouse.

Previous work in our laboratory showed that the osteopetrotic (op/op) mouse possesses a vitamin D-independent mechanism of intestinal calcium absorption. This study was performed in an effort to further characterize the mechanism. The vitamin D-deficient op/op mouse absorbed calcium faster than either a vitamin D-deficient or 1, 25-dihydroxyvitamin D(3)-supplemented wild-type mouse. This increased rate of absorption was not found at concentrations of calcium that result in diffusional calcium absorption. Thus, vitamin D-deficient op/op mice had intestinal calcium absorption similar to that of vitamin D-deficient wild-type littermates when increasing levels of calcium were administered. Also, mRNA and protein levels of calbindin-D9k were similar in vitamin D-deficient wild-type and op/op mice as well as in wild-type and op/op mice treated with 1, 25-dihydroxyvitamin D(3). Therefore, the mechanism of vitamin D-independent intestinal calcium absorption in the op/op mouse is distinct from vitamin D-dependent intestinal calcium absorption.

Evidence for involvement of 17beta-estradiol in intestinal calcium absorption independent of 1,25-dihydroxyvitamin D3 level in the Rat.

The sex steroid 17beta-estradiol (17beta-E2) has a broad range of actions, including effects on calcium and bone metabolism. This study with 3-month-old Brown Norway rats was designed to investigate the role of 17beta-E2 in the regulation of calcium homeostasis. Rats were divided in four groups, sham-operated, ovariectomized (OVX), and OVX supplemented with either a 0.025-mg or 0.05-mg 17beta-E2 pellet implanted subcutaneously. After 4 weeks, in none of the groups was serum calcium, phosphate, or parathyroid hormone altered compared with the sham group, while only in the OVX rats was a significant reduction in urinary calcium found. Bone mineral density and osteocalcin were modified, as can be expected after OVX and 17beta-E2 supplementation. OVX resulted in a nonsignificant increase in serum 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Supplementation with either one of the 17beta-E2 dosages resulted in an 80% reduction of 1,25(OH)2D3 and only a 20% reduction in 25-hydroxyvitamin D3 levels. OVX, as well as supplementation with 17beta-E2, did not affect serum levels of vitamin D binding protein. As a consequence, the estimated free 1,25(OH)2D3 levels were also significantly decreased in the 17beta-E2-supplemented group compared with the sham and OVX groups. Next, the consequences for intestinal calcium absorption were analyzed by the in situ intestinal loop technique. Although the 1,25(OH)2D3 serum level was increased, OVX resulted in a significant decrease in intestinal calcium absorption in the duodenum. Despite the strongly reduced 1,25(OH)2D3 levels (18. 1 +/- 2.1 and 16.4 +/- 2.2 pmol/l compared with 143.5 +/- 29 pmol/l for the OVX group), the OVX-induced decrease in calcium absorption could partially be restored by supplementation with either 0.025 mg or 0.05 mg of 17beta-E2. None of the treatments resulted in a significant change in calcium handling in the jejunum, although the trends were similar as those observed in the duodenum. 17beta-E2 did not change the VDR levels in both the intestine and the kidney. In conclusion, the present study demonstrates that 17beta-E2 is positively involved in intestinal calcium absorption, and the data strengthen the assertion that 17beta-E2 exerts this effect independent of 1,25(OH)2D3. In general, 17beta-E2 not only affects bone turnover but also calcium homeostasis via an effect on intestinal calcium absorption. (J Bone Miner Res 1999;14:57-64)

Defects in embryonic hindbrain development and fetal resorption resulting from vitamin A deficiency in the rat are prevented by feeding pharmacological levels of all-trans-retinoic acid.

Vitamin A is required for reproduction and normal embryonic development. We have determined that all-trans-retinoic acid (atRA) can support development of the mammalian embryo to parturition in vitamin A-deficient (VAD) rats. At embryonic day (E) 0.5, VAD dams were fed purified diets containing either 12 micrograms of atRA per g of diet (230 micrograms per rat per day) or 250 micrograms of atRA per g of diet (4.5 mg per rat per day) or were fed the purified diet supplemented with a source of retinol (100 units of retinyl palmitate per day). An additional group was fed both 250 micrograms of atRA per g of diet in combination with retinyl palmitate. Embryonic survival to E12.5 was similar for all groups. However, embryonic development in the group fed 12 micrograms of atRA per g of diet was grossly abnormal. The most notable defects were in the region of the hindbrain, which included a loss of posterior cranial nerves (IX, X, XI, and XII) and postotic pharyngeal arches as well as the presence of ectopic otic vesicles and a swollen anterior cardinal vein. All embryonic abnormalities at E12.5 were prevented by feeding pharmacological amounts of atRA (250 micrograms/g diet) or by supplementation with retinyl palmitate. Embryos from VAD dams receiving 12 micrograms of atRA per g of diet were resorbed by E18.5, whereas those in the group fed 250 micrograms of atRA per g of diet survived to parturition but died shortly thereafter. Equivalent results were obtained by using commercial grade atRA or atRA that had been purified to eliminate any potential contamination by neutral retinoids, such as retinol. Thus, 250 micrograms of atRA per g of diet fed to VAD dams (approximately 4.5 mg per rat per day) can prevent the death of embryos at midgestation and prevents the early embryonic abnormalities that arise when VAD dams are fed insufficient amounts of atRA.

Activity of various hydroxylated vitamin D3 analogs for improving phosphorus utilisation in chicks receiving diets adequate in vitamin D3.

1. Young chicks were used to evaluate the efficacy of 2 new vitamin D3 analogs relative to 1 alpha-hydroxycholecalciferol (1 alpha-OH D3) for their ability to improve the bioavailability of phytate-bound phosphorus (P) when added to P-deficient maize-soyabean meal diets that were superadequate in cholecalciferol. 2. Both 20-epi-19-nor-1,25-(OH)2 D3 and 20-epi-19-nor-1 alpha-OH D3 were observed to have phytate-P releasing activity, as measured by bone ash responses. 3. However, the bioactivity of the 2 new analogs differed when compared with 1 alpha-OH D3. The 20-epi-19-nor-1 alpha-OH D3 analog had a lower (P < 0.05) phytate-P releasing activity (45%) than either 1 alpha-OH D3 of 20-epi-19-nor-1,25-(OH)2 D3, which did not differ. 4. A dietary concentration of 10 to 15 micrograms/kg 1 alpha-OH D3 was found to optimize phytate-P utilisation in 2-week-old chicks.

1,25-dihydroxyvitamin D3 is a positive regulator for the two anti-encephalitogenic cytokines TGF-beta 1 and IL-4.

Previously we demonstrated that 1,25-dihydroxyvitamin D3 blocks the progression of relapsing encephalomyelitis. We now propose that 1,25-dihydroxyvitamin D3 blocks these autoimmune symptoms by stimulating the differentiation and/or function of cells that inhibit the encephalitogenic process. To support this belief, we have found that 1,25-dihydroxyvitamin D3 administration to mice increases IL-4 transcripts by 3- to 25-fold and TGF-beta 1 transcripts by 4- to 24-fold. Similarly, IL-4 and TGF-beta 1 transcripts were higher in the central nervous system of 1,25-dihydroxyvitamin D3-treated mice compared with controls. The number of cells recoverable from the lymph nodes of 1,25-dihydroxyvitamin D3-treated mice was only 50% that of controls. Overall, 1,25-dihydroxyvitamin D3 treatment causes a net loss in the total number of lymphocytes while the number of IL-4 and TGF-beta 1 transcripts increased. The systemic and local increase in the expression of these two anti-inflammatory cytokines by 1,25-dihydroxyvitamin D3 may be responsible for the ability of this drug to block encephalomyelitis.

Identification of a new all-trans-retinol metabolite produced through a new retinol metabolic pathway.

In vitro incubation of all-trans-retinol (atROL) with kidney homogenate from vitamin A-deficient and retinoic acid-supplemented (VAD-RAS) female rats produces a new retinol metabolite. Reverse-phase (RP) and normal-phase (NP) high-performance liquid chromatography (HPLC) analysis showed that this metabolite coelutes with the unknown all-trans-retinol (atROL) metabolite previously found in the day 10 conceptus and kidneys of vitamin A-deficient rats maintained on all-trans-retinoic acid (VAD-RA) and given 2 microg of [3H]atROL. Normal-phase (NP) HPLC purification of the metabolite collected from a RP HPLC column further separated the radiolabeled material into two components. The two isolated compounds have identical or very similar spectroscopic properties. Their nuclear magnetic resonance (1H NMR) and mass spectra (MS) indicated that they are isomers. Spectroscopic studies of the metabolites and their derivatives showed that they are nine-carbon fragments resulting from an oxidative cleavage of the side chain of atROL. The cleavage occurs at C-9, and the product is then oxidized to a keto group. The primary hydroxy group from atROL is preserved in the metabolite. A sulfide bridge is formed between C-11 and C-14, which interrupts the conjugation. The formation of the new metabolites, possessing a 2,5-dihydrothiophene ring, is catalyzed by an enzyme(s) located in the cytosolic fraction of kidneys. The process represents a new retinol metabolic pathway; however, its biological significance is unknown.

Cloning and expression of the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA.

Using a cDNA probe from the rat 24-hydrovitamin D3 24-hydroxylase, the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA has been isolated from a chicken kidney lambda gt11 library. The high degree of similarity with the mammalian 24-hydroxylase cDNAs strongly supports the belief that it is the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA. The deduced amino acid sequences are also very well conserved and 325 of them are identical among the four known 25-hydroxyvitamin D3 24-hydroxylases. This cDNA expressed in E. coli produces 24-hydroxylase activity.

1,25-Dihydroxycholecalciferol inhibits the progression of arthritis in murine models of human arthritis.

1,25-Dihydroxycholecalciferol [1,25-(OH)2D3] has been shown to inhibit the progression of experimental autoimmune encephalomyelitis (EAE). Here we tested the possibility that 1, 25-dihydroxycholecalciferol might be therapeutic for another autoimmune disease, arthritis. Two different animal models of arthritis were tested, namely, murine Lyme arthritis and collagen-induced arthritis. Infection of mice with Borrelia burgdorferi (the causative agent of human Lyme arthritis) produced acute arthritic lesions including footpad and ankle swelling. Supplementation with 1,25-dihydroxycholecalciferol of an adequate diet fed to mice infected with B. burgdorferi minimized or prevented these symptoms. Mice immunized with type II collagen also developed arthritis. The symptoms of this disease were also prevented by dietary supplementation with 1,25-dihydroxycholecalciferol. 1, 25-Dihydroxycholecalciferol given to mice with early symptoms of collagen-induced arthritis prevented the progression to severe arthritis compared with untreated controls. These results suggest that 1,25-dihydroxycholecalciferol and/or its analogs may be a valuable treatment approach to this disease.

Cloning and expression of rat 25-hydroxyvitamin D3-1alpha-hydroxylase cDNA.

A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D3-1alpha-hydroxylase (P4501alpha), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(+). When P4501alpha cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D3-1alpha-hydroxylase activity. The sequence analysis showed that P4501alpha was of 2,469 bp long and contained an ORF encoding 501 amino acids. The deduced amino acid sequence showed a 53% similarity and 44% identity to the vitamin D3-25-hydroxylase (CYP27), whereas it has 42.6% similarity and 34% identity with the 25-hydroxyvitamin D3-24-hydroxylase (CYP24). Thus, it composes a new subfamily of the CYP27 family. Further, it is more closely related to the CYP27 than to the CYP24. The expression of P4501alpha mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1alpha,25-dihydroxyvitamin D3 (rats fed a low Ca diet), P4501alpha mRNA was greatly increased in the renal proximal convoluted tubules.

Molecular cloning of cDNA and genomic DNA for human 25-hydroxyvitamin D3 1 alpha-hydroxylase.

The 25-hydroxyvitamin D3 1 alpha-hydroxylase (1 alpha-hydroxylase) is a cytochrome P450 enzyme that catalyzes the conversion of 25-hydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3. This enzyme plays an important role in calcium homeostasis. Here we report the molecular cloning of cDNA and gene for human 1 alpha-hydroxylase. The cDNA clone was obtained from a human kidney cDNA library by cross-hybridization with a previously cloned rat cDNA probe. The cDNA consists of 2469 bp and encodes a protein of 508 amino acids that shows 82.5% sequence identity with the rat enzyme. A computer-aided homology search revealed that 1 alpha-hydroxylase shares a relatively high homology with vitamin D3 25-hydroxylase (about 40% amino acid identity). Northern blot analysis showed that the 2.5-kb mRNA is most abundant in kidney. The gene for human 1 alpha-hydroxylase spans approximately 6 kb, is composed of nine exons, and is present as a single copy. This molecular cloning makes it possible to investigate the genetic mechanism of diseases related to calcium metabolism, including vitamin D-dependency rickets type I.

Cloning and characterization of the mouse vitamin D receptor promoter.

The gene encoding the mouse vitamin D receptor has been cloned. A new exon 1 has been found that changes the numbering established for the human VDR gene. Exons 2 and 3 in the human VDR gene (coding for the zinc fingers 1 and 2, respectively) are named exons 3 and 4 in the mouse vitamin D receptor. The 1.5-kb 5'-flanking region of the new exon 1 was analyzed and revealed the presence of putative cis-acting elements. Despite the absence of a TATA box, this 5'-flanking region contains several characteristics of a GC-rich promoter including four Sp1 sites present in tandem and two CCAAT boxes. Interestingly, the Sp1 site that is the most proximal to the new exon 1 overlaps a perfect site for Krox-20/24. Krox-20 is a transcription factor involved in brain development, and also in bone remodeling. In luciferase reporter gene expression assays, we showed that sequences from this 5'-flanking region elicit high transactivation activity. Furthermore, in the NIH 3T3 cell line, a 3- to 5-fold increase in response to forskolin treatment (an activator of adenylate cyclase and in turn of protein kinase A pathway) was observed.

Cloning and origin of the two forms of chicken vitamin D receptor.

The cDNA for the chicken vitamin D receptor (VDR) has been cloned in full length by screening cDNA libraries from chicken intestine and chicken kidney. The chicken kidney cDNA library constructed by both oligo(dT) and random primer methods yielded several full-length clones. These contained a 66-bp additional coding sequence at the 5' end in comparison to the cDNAs of human, rat, and mouse VDR. The Japanese quail VDR cDNA was also cloned in full length. We found that the 5' coding sequence reported previously had been artifactually inverted. Both chicken and quail cDNAs have three conserved ATG sites for translation initiation with the 3' one corresponding to the only ATG site found in the mammalian cDNAs. Northern blot analysis showed that the VDR gene is expressed as a single 2.69-kb transcript in chicken intestine and kidney. An analysis of the avian multiple ATG sites revealed that the first ATG is in a suboptimal context of TCCATGT, while the second ATG is in AGC-ATGG, matching the optimal context: R-3NNATGG+4. As a result, the two forms of chicken VDR (cVDR) (form A, 60.3 kDa; form B, 58.6 kDa) likely arise from the first and second ATG sites of a single mRNA during translation. Mutational analysis confirmed this belief. The wild-type construct starting from the first ATG site with its original context was subcloned into pCMV5 expression vector and expressed in COS-1 cells. Two receptor proteins, exactly comigrating with the form A and form B cVDRs isolated from chicken intestine, were detected by immunoblotting. Point mutations optimizing the first ATG context led to exclusive production of form A, while knocking out the first ATG site resulted in the sole generation of form B. Form A translated from the first ATG site has 451 amino acids, and form B from the second ATG has 437 amino acids. Comparison between avian and mammalian VDRs indicated that VDR is essentially conserved in DNA binding and ligand binding domains. The significant difference especially at the N-terminus demonstrates divergence of this receptor during evolution of these species.

Retinol is specifically required during midgestation for neonatal survival.

Previous work has demonstrated that vitamin A-deficient, retinoic acid-supplemented pregnant rats cannot complete gestation without the administration of retinol. As little as 2 micrograms administered on day 10 of gestation is sufficient to prevent the characteristic fetal resorption that begins at day 15 of gestation. This single dose of retinol supports continued development through day 20 of gestation. However, if gestation is allowed to proceed to parturition, the newborn pups die within a few minutes of being severed from the umbilical cord. The pups are born with a pink and healthy skin tone, but within seconds of umbilical separation, they begin to gasp for air, become cyanotic in appearance, and die within several minutes from an apparent inability to obtain oxygen. Histological examination of these neonates demonstrates delayed pulmonary development. Branching and scalloping of ducts and saccule and subsaccule formation are decreased. This phenotype is consistent with that observed in respiratory distress syndrome seen in some premature human infants.

Hypophosphatemia and the development of rickets in osteopetrotic (op/op) mice.

Our previous work has shown that op/op mice hyperabsorb dietary calcium in the vitamin D-deficient state and shunt that calcium into bone. Under these conditions, the op/op mice are hypocalcemic. The purpose of this study was to examine calcium metabolism and bone mineralization in vitamin D-deficient op/op mice. First, the op/op mice and their normal littermates were placed on a vitamin D-deficient, low phosphorus diet to limit bone mineralization. Under these circumstances, op/op mice survived, even when calcium was also removed from the diet. If the diet contained phosphate, op/op mice died from hypocalcemic tetany when calcium was also removed from the diet. Furthermore, serum calcium levels became similar to wild type in the op/op mice administered the vitamin D-deficient, low phosphorus diet, and op/op mice were able to increase serum calcium in response to 1,25-dihydroxyvitamin D3. The op/op mice developed rickets when their serum phosphorus level was too low to support bone mineralization. The op/op mice became hypophosphatemic on regimens in which normal mice were able to maintain normal serum phosphorus levels. It appears that the op/op mouse simply requires a higher dietary calcium and phosphorus level to prevent rickets and hypocalcemic tetany since the bone is not available as a source of these minerals. However, the ability of the op/op mouse to mineralize bone at low serum calcium and phosphorus levels remains unexplained.