Electrochemical regeneration of granular activated carbons loaded with phenol and natural organic matter.

The main objective of this study was to assess the performance of electrochemical regeneration of granular activated carbon via a set of bench-scale experiments using different operating conditions in the regeneration of several different activated carbons loaded with phenol or natural organic matter. The regeneration efficiency can be increased by increasing the charge applied, whether this was achieved by an increase in current or regeneration time. The degree of phenol-adsorption saturation did not significantly affect the regeneration efficiencies. The regeneration efficiencies of the various types of phenol-loaded activated carbon were quite similar despite the differences in their conductivity. The activated carbon exhibiting fully reversible adsorption of phenol had slightly higher regeneration efficiencies than those involving partially irreversible adsorption. Electrochemical regeneration of activated carbon is feasible at a laboratory scale as regeneration efficiencies up to 80% were achieved during electrochemical regeneration of phenol-loaded or natural organic matter-loaded activated carbons.

K depletion and NH4Cl acidosis increase apical insertion of studded membrane in type A intercalated cells.

To examine the effects of K depletion and metabolic acidosis on the prevalence and distribution of H(+)-adenosinetriphosphatase (H(+)-ATPase)-related studs at the luminal plasma membrane of A-type intercalated cells (A-ICs), we conducted a quantitative electron microscopical study on the cortical collecting ducts (CCDs) of control, NH4Cl-loaded, and K-depleted rats. The percentage of A-ICs was slightly increased in the K-depleted but not in the acidotic rats. A-ICs were considered "active" when they presented a semicontinuous row of 9- to 10-nm studs at the cytoplasmic face of the apical membrane and "inactive" when all of the apical membrane was devoid of studs. The percentage of active A-ICs was greatly increased in acidotic (87.2%) and K-depleted (79.3%) rats compared with controls (41.6%). These results give a quantitative expression to the general view that acidosis elicits insertion of studded membrane in the apical plasma membrane of A-ICs. Furthermore, we show, for the first time, that an increase in the membrane insertion of H(+)-ATPase is also part of the response to K depletion.

Detection of different adenosine triphosphatases in human placental brush border membranes.

The microvillous membrane of human placental syncytiotrophoblast cells contains a high ATPase activity. The purpose of this study was to characterize this activity and to investigate the presence of vacuolar type H+ ATPase in this membrane. Intact brush border membrane vesicles strongly hydrolyzed ATP, reflecting the presence of ATPase on the external side of the membrane. The ATPase activity was entirely Mg2+ dependent and increased with pH. At pH 7.5, Vmax was 31.0 +/- 1.7 mumol/mg/20 min and Km 0.18 +/- 0.03 mM ATP. Hydrolysis of ATP was not influenced by the presence of bicarbonate or alkaline phosphatase inhibitors, but at pH 8 it decreased by half following addition of 100 microM dicyclohexylcarbodiimide (DCCD). At pH 7.5, 1 mM N-ethylmaleimide (NEM) depressed this activity by less than 5%. Opening the membrane vesicles with 0.1% desoxycholate (DOC) or Triton-X neither revealed any additional ATPase activity nor altered the low sensitivity to NEM. Treatment of these membranes with 1% cholate decreased the ATPase activity by more than 70% and did not enhance the sensitivity of ATP hydrolysis to NEM. 10(-7) M Bafilomycin, which reduced by 56 +/- 9% the ATPase activity in dog kidney brush border membranes treated with 0.1% DOC, had no effect on placental brush border membranes subjected to the same procedure. Finally, neither immunocytochemical staining using monoclonal antibody to the M(r) 31000 subunit of V-type H+ ATPase, nor electron microscopic examination detected the presence of H(+) ATPase in placental membranes. In conclusion, the placental brush border membrane is the site of a strong "ecto" ATPase activity which is partially DCCD sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and immunochemistry of a soluble matrix protein of the chicken eggshell (ovocleidin 17).

The protein components of biomineralized structures (matrix proteins) are believed to modulate crystal nucleation and growth, and thereby influence the shape and strength of the final structure. The chicken eggshell contains a complex array of distinct matrix proteins. The most abundant of these was purified to homogeneity by a combination of anionic exchange and hydroxyapatite chromatographies. Antibodies to this protein were raised in rabbit, and utilized for Western blotting and immunohistochemistry. These studies indicated that the 17 kDa antigen (ovocleidin 17, OC-17) is found in the shell gland mucosa, and that only the tubular gland cells were positive. Immunohistochemistry with decalcified shell indicated that OC-17 is uniformly distributed throughout the shell matrix, but concentrated in the mammillary bodies. Our results indicate that this protein is secreted during shell formation and becomes incorporated into this structure. It may therefore play a role in the crystallization process and influence the properties of the resulting eggshell.

Ultrastructural and immunocytochemical evidence for the presence of polarised plasma membrane H(+)-ATPase in two specialised cell types in the chick embryo chorioallantoic membrane.

The chick embryo, confined in the eggshell, has to dispose/buffer the acid generated by its metabolism, as well as to release calcium from the shell which is used for growth. To localise H(+)-ATPase, electron microscope and immunocytochemical studies were conducted on chorioallantoic membranes of 15-17 d chick embryos. Ultrastructural studies of the villus cavity (VC) cells in the chorionic epithelium demonstrated that their apical plasma membrane, juxtaposed with the shell membranes, contains microvilli as well as microplicae which possess 9-10 nm studs at a density of 16,700 particles/micron2, a characteristic feature of the polarised H(+)-ATPase pump. Immunocytochemical staining, using a monoclonal antibody to the 31 kDa subunit of H(+)-ATPase, confirmed the presence of large amounts of the vacuolar H(+)-ATPase in the VC shells with a distribution highly polarised towards the eggshell membranes. Immunoelectron-microscopic localisation studies using a rabbit antiserum to whole bovine H(+)-ATPase and immunogold technique, confirmed the localisation of H(+)-ATPase at the apical microvilli/microplicae as well as in the subapical vesicles. In the allantoic epithelium, the presence of mitochondria-rich (MR) cells was confirmed; it was shown that these cells extend through the full thickness of this epithelium. The MR cells also contained large numbers of 9-10 nm studs, typical of proton secreting cells, in their apical plasma membrane. This was confirmed by immunocytochemical staining which showed abundant localisation of H(+)-ATPase in these cells; this localisation was, however, diffuse rather than apical.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of intercalated cell changes in rat pups from acid- and alkali-loaded mothers.

We evaluated the effects of acid-base changes in pregnant and lactating dams on intercalated cell morphology and populations in newborn and 2-wk-old rats. Collecting ducts were studied with transmission electron microscopy with intercalated cells identified by the presence of 10-nm studs in the cytoplasmic face of apical membranes (A-type intercalated cells) or basolateral membranes (B-type intercalated cells). In newborn and 2-wk-old pups from dams with metabolic alkalosis, there was a significantly larger percentage of B-type intercalated cells and a smaller percentage of A-type intercalated cells. Acid loading with NH4Cl, however, did not produce an increase in the percentage of A-type intercalated cells, but reduced the percentage of B-type intercalated cells. We conclude that maternal metabolic alkalosis is associated with an increase in the percentage of B-type intercalated cells, suggesting that the initial differentiation of intercalated cells is responsive to maternal acid-base disturbances.

Soluble protein constituents of the domestic fowl's eggshell.

1. The protein components of the domestic fowl's eggshell are believed to influence appreciably the mechanical properties of the shell and/or its biomineralisation. The purpose of this study was to compare the protein species composing the eggshell matrix in different parts of the shell structure, by SDS-PAGE and chromatography, utilising eggshell cleaned by different methodologies. 2. Protein species were identified whose absence was associated with the removal of the mammillary knobs. In particular, a prominent 81 kDa protein, as well as 38 and 54 kDa calcium-binding proteins, were concentrated within the mammillary layer, as was a 129 kDa insoluble protein. By contrast, soluble proteins of 54, 33, 22, and 14 kDa were enriched in the palisade layer. 3. Our results demonstrate that the mineralised layers of the fowl's eggshell possess a complex array of distinct proteins. The different proteins which have been detected in the mammillary and palisade layers may be related to the distinct crystallisation patterns of calcium carbonate in these zones of the eggshell.

Effects of excess iodine in chick embryo thyroid follicles: initial inhibition and subsequent hypertrophy.

The effects of excess iodine on the development of the thyroid gland of chick embryos was assessed following injections of potassium iodide prior to incubation. Iodide injection resulted in a significantly greater thyroid gland weight (goitre) on Day 18 of incubation and a delay in hatching. Histological studies of the thyroid gland on Day 12 of incubation revealed that iodide injection had inhibited thyroid follicle development. On Day 14, however, the thyroid glands of the iodide-treated embryos were indistinguishable from controls and on Day 18 the thyroid follicles of the iodide-injected embryos were clearly hypertrophied. In agreement with these light microscopical observations, electron microscopical examination showed conspicuous development of rough endoplasmic reticulum in the follicle cells of both iodide-treated 14 and 18 days old embryos and in those of the corresponding controls. Immunocytochemical studies of the pituitary of 18 days old embryos revealed a depletion of immunoreactive TSH suggesting that the iodide-induced hypertrophy of the thyroid was mediated by an activation of the thyrotropes. Iodide treatment was without effect on plasma levels of T3 and T4 for Day 18 embryos suggesting that the compensatory hypertrophy of the thyroid gland was sufficient to maintain circulating levels of thyroid hormones. The present results demonstrate that, in the embryonic chick thyroid, excess iodine produces effects which occur in two phases. The first phase consists of a transitory inhibition of the formation of follicles; it is followed by a second phase of compensatory hypertrophy resulting in goitre. The first phase probably results from a direct inhibitory effect of iodine on the developing thyroid whereas the second phase probably reflects a stimulation of the thyroid by TSH.

Differentiation of renal intercalated cells in fetal and postnatal rats.

An ultrastructural study was conducted on the kidneys from rat fetuses and pups from ages ranging from birth to 8 weeks to identify the time of appearance of each of the two intercalated cell types. With transmission electron microscopy. A-intercalated cells were recognized by their large apical microvilli and microplicae as well as by the numerous subapical vesicles. Their identification was confirmed by the presence of typical studs at the cytoplasmic face of the apical plasma membrane. By scanning electron microscopy the cells were recognized by their typical microplicae at the apical surface. In 19-day-old fetuses and newborns. A-intercalated cells were numerous in the epithelium lining the renal pelvis and inner medullary intercalated ducts. Two weeks after birth they disappeared from these regions but became numerous at the outer medullary collecting ducts and also at the cortical collecting ducts although to a lesser degree. B-intercalated cells were recognized by the scarcity of microvilli, the absence of microplicae, and the large number of basal infoldings. Their identification was confirmed by the presence of studs at the cytoplasmic face of the basolateral membrane. B-cells started to appear 3 weeks after birth and increased thereafter. We speculate that the particular stages at which the two cell types differentiate might be related to changes in acid-base status.

Optimal dietary level of 1 alpha,25-dihydroxycholecalciferol for eggshell quality in laying hens.

The optimal dietary level of 1 alpha,25-dihydroxycholecalciferol [1,25-(OH)2D3] for eggshell quality was established. White Leghorn hens, 59 wk of age, were fed one of eight diets that contained the same basal ingredients, including 3.1% calcium, but different levels (microgram/kg) or forms of calciferol supplements: no calciferol supplement of any form (56 hens); 27.5 (control) or 55.0 micrograms of cholecalciferol (56 hens each); 3, 5, or 7 micrograms of 1,25-(OH)2D3 (28 hens each); 5 micrograms of 24,25-dihydroxycholecalciferol [24,25-(OH)2D3] with 28 hens; 5 micrograms each of 1,25-(OH)2D3 and 24,25-(OH)2D3 (28 hens). All groups were fed the control diet prior to the 21-wk treatment. The group fed 5 micrograms 1,25-(OH)2D3/kg diet ranked first in specific gravity (SG), e.g., 1.081 versus 1.077 for the control group at Week 21 (P less than .05). The group fed 7 micrograms 1,25-(OH)2D3/kg consumed 30% less feed and laid 20% fewer eggs than the control, but shell quality was not affected. The groups receiving no calciferol supplement or receiving only 24,25-(OH)2D3 laid eggs with significantly lower SG than the control after 2 wk of treatment (1.072 or less versus 1.082 at Week 2). The rest of the treatment groups mentioned were comparable to the control in eggshell quality and egg production. Groups fed the combination of 1,25-(OH)2D3 and 24,25-(OH)2D3 per kilogram of feed, or 1,25-(OH)2D3 alone at 5 micrograms/kg, had significantly higher tibial weights relative to the control group. All groups receiving the diets without cholecalciferol supplementation had markedly reduced hatchability. It was concluded that the optimal dietary level of 1,25-(OH)2D3 for improving eggshell quality without affecting egg production was approximately 5 micrograms/kg and the toxic level was 7 micrograms/kg.

Effects of vitamin D3 metabolites on physiological traits of White Leghorn hens.

This study was conducted to determine the effect of feeding vitamin D3(D3) metabolites on BW of hen, weight of uterus, plasma Ca, jejunal and uterine adenosine triphosphatase (ATPase), and carbonic anhydrase. At 416 days of age each of 7 groups of laying hens was fed the basal ration supplemented with one of 7 concentrations (micrograms per kg) of D3 or its metabolites as treatments: 0 micrograms of D3; 27.5 micrograms of D3; 3, 5, or 7 micrograms of 1,25(OH)2D3; 5 micrograms of 24,25(OH)2D3; and 5 micrograms of 24,25(OH)2D3 plus 5 micrograms of 1,25(OH)2D3. Treatment effects were compared at various periods after the start of the study. Hens fed the unsupplemented ration had lower (P less than .05) values for all traits than hens fed the D3-supplemented ration by 162 days after the start of treatment. In a comparison of all dietary treatments except the one involving 0 micrograms D3, from 154 to 161 days after the start of the experiment, treatment effects were significant (P less than or equal to .05) for BW, uterine ATPase, and carbonic anhydrase; hens fed 5 micrograms of 24,25(OH)2D3 per kg of ration ranked the lowest of all treatment groups for these traits. Hens fed 27.5 micrograms of D3 and those fed 5 micrograms of 1,25(OH)2D3 per kg of ration did not differ (P greater than .05) for any traits studied. The results suggest that 5 micrograms of 1,25(OH)2D3 per kg of ration can replace 27.5 micrograms of D3 per kg of ration but that 5 micrograms of 24,25(OH)2D3 per kg of ration tends to have a negative effect on physiological systems of the hen.

Effect of 1 alpha,25-dihydroxycholecalciferol on egg shell quality and egg production.

1. The effect of replacing dietary cholecalciferol (D3) by 1 alpha,25-dihydroxycholecalciferol (1,25-(OH)2D3) on egg shell quality and egg production was tested on 32-week-old White Leghorn laying hens over 9 weeks. 2. Hens fed on a diet supplemented with 5 micrograms 1,25-(OH)2D3/kg diet, tended to lay more eggs, and the eggs had significantly higher specific gravity and percentage shell than eggs from control hens fed on a diet supplemented with 27.5 micrograms D3/kg diet. 3. The effect became apparent after about 4 weeks of treatment and persisted until the end of the test. 4. Hens fed on a diet without D3 supplement started to lay very thin or soft shelled eggs within 4 weeks, suggesting that the birds' reserves of D3 or its metabolites were depleted within this period. 5. The results suggest that 1,25-(OH)2D3 can be substituted for D3 in layer diets to improve egg shell quality.

Vitamin D deficiency in the chick embryo: effects on prehatching motility and on the growth and differentiation of bones, muscles, and parathyroid glands.

Vitamin D-deficient chicken embryos were obtained by feeding laying hens diets in which 3-7 micrograms calcitriol replaced the vitamin D3 supplement. A large proportion of the D-deficient embryos failed to complete the prehatching positional changes required to start pulmonary respiration. For this reason most of them became cyanotic and had subcutaneous edema and hemorrhages in the head and neck and died without hatching. Total as well as leg-bone and muscle weights were significantly lower in the deficient embryos than in the controls and these changes probably explain the inability of the embryos to complete the movements required to place the beak in contact with the air chamber and start pulmonary respiration. The histological study of the tibiae showed decreased mineralization with narrower trabeculae and enlarged osteoid seams; bone resorption at the inner surface was also significantly decreased. The ultrastructural study of parathyroid glands showed increased functional activity reflected by increased number and size of cisternae of rough endoplasmic reticulum. Injection of 10 ng calcitriol, 1 microgram 24,25-(OH)2D3, or 2 micrograms 25OHD3 to deficient embryos on the 14th day of incubation improved hatchability, bone and muscle weights, and both bone mineralization and resorption.

Role of calcitriol in phosphate regulation by the chick embryo.

Chick embryos were injected on the 14th day of incubation with 100 ng calcitriol. The concentration of Ca in their serum rose significantly 4 hours after the injection and the concentration of Pi started to decrease 10 hours after. When embryos of the same age were injected with a solution containing CaCl2, the concentrations of both Ca and P rose significantly 2 hours after the injection and remained high until the end of the experiment. The fact that both treatments produced hypercalcemia but had opposite effects on the concentration of Pi does not agree with the idea that the hypophosphatemic response to calcitriol might be secondary to the hypercalcemia which precedes it. The injection of a solution of NaHCO3 to embryos of the same age failed to produce hypophosphatemia. The fact that calcium salts and bicarbonate, when injected separately, fail to induce hypophosphatemia does not contradict the possibility that the hypophosphatemic response to calcitriol might result from the simultaneous increase in flux of Ca and -HCO3 from the shell. Three days after the injection of calcitriol to 14-day-old embryos, the total amount of Ca and P in the urine was significantly higher than in the controls. The concentration of Ca and P in kidney tissue was also significantly higher in the injected embryos. In addition, calcified precipitates were detected histochemically in the lumen of the kidney tubules from the treated embryos. These results are interpreted as demonstrating that an increase in the excretion of P in the urine is the main mechanism explaining calcitriol-induced hypophosphatemia in the chick embryo.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cholecalciferol or calcium deficiency on oestrogen metabolism in the laying hen.

1. Seventeen 32-week-old White Leghorn laying hens were induced to become deficient in calciferol or in calcium (laying thin or soft shelled eggs) by withdrawing either cholecalciferol (27.5 micrograms/kg diet) or calcium (31 g/kg diet) supplements from the control diet. 2. The metabolic fate and metabolic clearance rate (MCR) of intravenously injected 3H-oestradiol-17 beta were then monitored for 40 min. 3. In both the calciferol and calcium-deficient groups a major oestrogen sulphate pathway was particularly affected, resulting in a decreased conversion of oestradiol-17 beta-3-sulphate to oestradiol-17 alpha-3-sulphate, with a concomitant reduced MCR of oestradiol-17 beta from plasma. 4. The metabolic defect was corrected by feeding the control diet. 5. Because the metabolic defect observed in calciferol deficiency occurred in Ca deficiency in a more severe form, we conclude that the more immediate cause was calcium rather than calciferol deficiency. To our knowledge, this is the first observation of a calcium-deficient effect on oestrogen sulphate metabolism in vivo.

Experimental induction of microphthalmia in the chick embryo with a single dose of cisplatin.

Chick embryos were injected on the fifth day of incubation with 75 ng cis-diamminedichloroplatinum II (cisplatin) and killed at daily intervals. Bilateral microphthalmia appeared in 88% of the surviving embryos; the decrease in eye size was noticeable 2 or 3 days after injection. Coinciding with this, macroscopic, histological, and ultrastructural changes started to appear in the ciliary body: ciliary processes failed to form and the cells in the inner layer of the ciliary epithelium underwent degenerative changes. Changes in the retina appeared somewhat later. Despite the decreased growth rate of the whole eye the neural layer of the retina continued to grow rapidly; as a result, it formed numerous folds and acquired a glandular appearance. In the most severe cases the rapidly growing retina would invade the ciliary region and replace completely the degenerated inner layer of the ciliary epithelium. It has been shown by previous authors that intraocular pressure is a determinant of eye expansion and also that the secretion of water and ions by the ciliary epithelium is important for the maintenance of that intraocular pressure. On this basis, our results are interpreted as indicating that the primary lesion induced by cisplatin was in the ciliary epithelium and that microphthalmia was the consequence of decreased pressure. It is also concluded that the retinal changes were due to the fact that the retina continued to grow despite the lack of expansion of the eye as a whole.

Scanning electron microscopy of thin and soft shells induced by feeding calcium-deficient or vitamin D-deficient diets to laying hens.

A scanning electron microscopic study was conducted on shells from eggs laid by four groups of hens maintained on different types of diets: a) control, b) vitamin D3-deficient, c) Ca-deficient, and d) vitamin D3-deficient supplemented with 1,25-(OH)2D3. After 1 week for Ca-deficient hens and after 4 weeks for vitamin D3-deficient hens, the thickness of the shell decreased abruptly and numerous thin-shelled and soft-shelled eggs were laid. The study showed that with both Ca-deficient and vitamin D3-deficient diets, the outer layers of the shell (cuticle and spongy) were reduced or absent but the mammillary layer was present even in the thinnest soft-shelled egg. The order in which layers disappeared as treatment progressed was exactly the reverse of the order in which these layers are formed in normal eggs. No eggs were found without mammillary knobs, which suggests that the hens stop laying before Ca concentrations in blood become too low for the formation of the mammillary knobs. Uncalcified portions of the shell organic matrix were never found, suggesting that Ca deposition and matrix formation were inhibited simultaneously. The relationship between fibers of the shell membrane and mammillary knobs was preserved in all cases. The eggshells from hens on 1,25-(OH)2D3-supplemented diets were ultrastructurally indistinguishable from those of hens on diets adequate in vitamin D3.

Effects of vitamin D deficiency in the chicken embryo.

Vitamin D-deficient chicken embryos were obtained by feeding laying hens a diet in which 5 micrograms 1,25(OH)2D3/kg feed were substituted for the vitamin D3 supplement in the control diet. Hatchability, total Ca and inorganic P concentration in blood, and tibial ash/dry weight ratio were determined in the vitamin D-deficient embryos and in embryos obtained from hens fed the control diet supplemented with 1100 IU vitamin D3/kg feed. After 5 weeks on the substituted diet the hens laid eggs that showed decreased hatchability in spite of excellent shell quality. All determinations in blood and bones were made on embryos of eggs laid after 6-12 weeks on the diets. On the 17th day of incubation the embryos derived from hens fed the substituted diet showed significant hypocalcemia and hyperphosphatemia and a low tibial ash/dry weight ratio. Injection of 1,25(OH)2D3 3 days before killing corrected the hypocalcemia of the deficient embryos. Those chicks that managed to hatch had normal levels of calcium and inorganic phosphate 1 day after hatching. These findings support previous suggestions by us and other authors that vitamin D metabolites are required by the embryo in order to mobilize calcium from the shell, and decreased hatchability in vitamin D-deficient embryos is related to a defect in calcium mobilization from the shell. While in previous studies a decrease in hatchability was the only parameter used to judge D deficiency of the embryos in our present studies, the deficiency is confirmed by demonstrating a deficit in mineral metabolism which is a more specific sign of D deficiency.

Role of vitamin D in the development of the chick embryo.

Chick embryos of various ages and 1-day-old chicks were injected with different doses of 1,25(OH)2D3 and the concentrations of Ca and Pi in their blood were determined 24 hr after. It was confirmed that the dose required to induce hypercalcemia and hypophosphatemia was much lower in the case of 15-day-old embryos than in younger ones. The age of maximal response corresponds to the period when the chorionic epithelium is fully differentiated. Newly hatched chicks did not respond even with doses 20 times higher than those used in 15-day-old embryos. These findings support the idea that the humoral changes produced by 1,25(OH)2D3 result from increased resorption of calcium from the shell and that the chorionic epithelium is instrumental in this phenomenon. The autoradiographic study of tissues from embryos injected with tritiated 1,25(OH)2D3 showed that the exposure time required to detect nuclear concentration of radioactivity was much shorter in the case of "villus-cavity" cells than in any other tissue (kidney, intestine, bone, parathyroid glands) in which target cells were recognized. The above results are interpreted as indicating that the chorionic epithelium is the main target for 1,25(OH)2D3. The histological study of various organs from embryos injected with large doses of 1,25(OH)2D3 showed generalized infiltration of spleen, liver, and bursa of Fabricius with eosinophilic granulocytes. It has been suggested that 1,25(OH)2D3 influences the proliferation and differentiation of leukocytes; the chick embryo might be a good model for further exploration of this problem.

The role of ultimobranchial bodies in the modulation of the response of chick embryos to 1,25-dihydroxy-cholecalciferol.

Ultimobranchial bodies (UBBs) were dissected from 17-day-old chick embryos and grafted onto the chorioallantoic membrane of 8-day-old embryos. The embryos with UBB grafts as well as sham-grafted controls were injected on the 10th day of incubation with 100 ng 1,25(OH)2D3 dissolved in ethyl alcohol or with an equal volume of ethyl alcohol alone; embryos were sacrificed on the 13th day. Grafted UBBs showed ultrastructural characteristics typical of actively secreting glands. A histological study of the tibiae from all embryos showed that while the grafted embryos responded to the injection of 1,25(OH)2D3 with a peripheral rim of undermineralized bone trabeculae, sham-grafted embryos never did so. These results confirm the original hypothesis that the presence of differentiated UBBs is a precondition for the production of undermineralized bone (osteoid) by 1,25(OH)2D3. In a second series of experiments, similarly treated embryos were sacrificed on the 10th, 11th, 12th and 13th day; the levels of calcium and inorganic phosphate were determined in their blood. The injection of 1,25(OH)2D3 produced in all embryos hypercalcaemia and hypophosphataemia. However, the hypophosphataemic response was more prolonged in the embryos with UBB grafts than in sham-grafted ones. These results suggest that the grafted UBBs prolonged the hypophosphataemic response, probably by secreting calcitonin and thus reducing the rate of bone resorption. It is also probable that the prolonged hypophosphataemia produced or contributed to the undermineralization of the peripheral (subperiosteal) trabeculae.