Chick Embryo Bone Research Articles

Dexamethasone interferes with osteoblasts formation during osteogenesis through altering IGF-1-mediated angiogenesis.

Dexamethasone (Dex), a synthetic glucocorticoid (GC) with long-lasting treatment effects, has been proved to exert a modulatory effect on osteoblast proliferation and differentiation during embryonic osteogenesis. However, it is still controversial if Dex exposure influences endochondral ossification and the underlying mechanism. In this study, chick embryos in vivo and preosteoblast cell cultures in vitro were utilized to investigate the effects of Dex on osteoblast formation and differentiation during the skeletal development. We first demonstrated that Dex exposure could shorten the long bones of 17-day chick embryos in vivo, and also downregulated the expressions of osteogenesis-related genes. Next, we established that Dex exposure inhibited the proliferation and viability of preosteoblasts-MC3TC-E1 cells, and the addition of insulin-like growth factor 1 (IGF-1) could dramatically rescue these negative effects. On the basis of remarkable changes in the rescue experiments, we next verified the important role of angiogenesis in osteogenesis by culturing isolated embryonic phalanges in Dulbecco's modified Eagle's medium culture or on the chick chorioallantoic membrane (CAM). Then, we transplanted MC3T3-E1 cell masses onto the CAM. The data showed that Dex exposure reduced the vessel density within the developed cell mass, concomitantly with the downregulation of IGF-1 pathway. We verified that the inhibition of blood vessel formation caused by Dex could be rescued by IGF-1 treatment using the CAM angiogenesis model. Eventually, we demonstrated that the shortened length of the phalanges in the presence of Dex could be reversed by IGF-1 addition. In summary, these findings suggested that the inhibition of Igf-1 signal caused by Dex exposure exerts a detrimental impact on the formation of osteoblasts and angiogenesis, which consequently shortens long bones during osteogenesis.

Biphasic influence of dexamethasone exposure on embryonic vertebrate skeleton development

Dexamethasone (Dex) has anti-inflammatory and immunomodulatory properties against many conditions. There is a potential teratogenic risk, however, for pregnant women receiving Dex treatment. It has been claimed that Dex exposure during pregnancy could affect osteogenesis in the developing embryo, which still remains highly controversial. In this study, we employed chick embryos to investigate the effects of Dex exposure on skeletal development using combined in vivo and in vitro approach. First, we demonstrated that Dex (10−8–10−6μmol/egg) exposure resulted in a shortening of the developing long bones of chick embryos, and it accelerated the deposition of calcium salts. Secondly, histological analysis of chick embryo phalanxes exhibited Dex exposure inhibited the proliferation of chondrocytes, increased apoptosis of chondrocytes and osteocytes, and led to atypical arranged hypertrophic chondrocytes. The expression of genes related to skeletogenesis was also analyzed by semi-quantitative RT-PCR. The expression of ALP, Col1a2 and Col2a1 was decreased in the Dex treated phalanxes. A detectable increase was observed in Runx-2 and Mmp-13 expression. We next examined how Dex affected the different stages of skeletogenesis in vitro. Utilizing limb bud mesenchyme micromass cultures, we determined that Dex exposure exerted no effect on apoptosis but impaired chondrogenic cell proliferation. Interestingly, low dose of Dex moderately prompted nodule formation as revealed by alcian blue staining, but higher doses of Dex significantly inhibited similar chondrogenic differentiation. Dex exposure did not induce apoptosis when the chondrogenic precursors were still at the mesenchymal stage, however, cell viability was suppressed when the mesenchyme differentiated into chondrocytes. Alizarin red staining revealed that the capacity to form mineralized bone nodules was correspondingly enhanced as Dex concentrations increased. The mRNA level of Sox-9 was slightly increased in mesenchymal cell mass treated by low concentration of Dex. Mmp-13 expression was obviously up-regulated by Dex in both mesenchymal cells and primary chondrocyte cultures. And Col10a1 expression was also increased by Dex exposure in chondrocyte. In summary, we have revealed that different concentrations of Dex exposure during early gestation could exert a biphasic effect on vertebrate skeletal development.

Distribution of lovastatin to bone and its effect on bone turnover in rats

Statins, the widely used lipid-lowering drugs, are inhibitors of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, which catalyses a rate-limiting step in the biosynthesis of cholesterol. Many previous reports show that statins can act both as bone anabolic and as anti-resorptive agents but their beneficial effects on bone turnover are still controversial. Considering their high liver specificity and low oral bioavailability, the distribution of statins to the bone microenvironment is questionable. In this study, the distribution of lovastatin and its active metabolites to bone, with respect to plasma and liver compartments, was examined after oral and intravenous administration in female rats. As compared with oral administration, the distribution of lovastatin to the bone compartment was significantly enhanced after intravenous administration. Further, the effect of lovastatin on bone turnover was studied in-vitro and in-vivo to assess its anti-osteoporotic potential. Lovastatin acid but not lovastatin was found to inhibit parathyroid-hormone-induced bone resorption in an in-vitro chick embryo bone assay. Oral, as well as intravenous, short-term lovastatin treatment significantly reduced the serum total cholesterol, serum total alkaline phosphatase and urinary crosslinks in ovariectomized rats. In accordance with its increased distribution to the bone compartment, intravenously administered lovastatin was more effective in reducing the ovariectomy-induced increase in markers of bone metabolism, especially urinary crosslinks. The findings of this study suggest that statins inhibit bone resorption and that their anti-resorptive efficacy can be increased by administering them by routes other than oral so as to achieve their enhanced concentration in bone.

In vitro differentiation of chick embryo bone marrow stromal cells into cartilaginous and bone-like tissues.

Bone marrow stromal cells, progenitor cells involved in repair of bone and cartilage, can potentially provide a source for autologous skeletal tissue engineering. We investigated which factors were required to induce in vitro differentiation of avian bone marrow stromal cells into three-dimensional cartilaginous and bone-like tissues. Bone marrow stromal cells from embryonic chicks were expanded in monolayers, seeded onto biodegradable polyglycolic acid scaffolds, and cultured for 4 weeks in orbitally mixed Petri dishes. Cell-polymer constructs developed an organized extracellular matrix containing glycosaminoglycans and collagen, whereas control bone marrow stromal cell pellet cultures were smaller and consisted predominantly of fibrous tissue. Bone marrow stromal cells expanded with fibroblast growth factor-2 and seeded onto polymer scaffolds formed highly homogeneous three-dimensional tissues that contained cartilage-specific molecular markers and had biochemical compositions comparable with avian epiphyseal cartilage. When cell-polymer constructs were cultured in the presence of beta-glycerophosphate and dexamethasone, the extracellular matrix mineralized and bone-specific proteins were expressed. Our work shows that cell expansion in the presence of fibroblast growth factor-2 and cultivation on a three-dimensional polymer scaffold allows differentiation of chick bone marrow stromal cells into three-dimensional cartilaginous tissues. In the in vitro system studied, the same population could be selectively induced to regenerate either cartilaginous or bone-like tissue.

Stimulative effects of gusuibu (Drynaria baronii) injection on chick embryo bone primordium calcification in vitro.

Through tissue culture and isotope tracing, it was found that Gusuibu (Drynaria baronii) injection (GI) significantly promoted calcification of the cultivated chick embryo bone primordium (CEBP), increased ALP activity in the cultivated tissue, and accelerated synthesis of proteoglycan. It was also confirmed that the promotion of proteoglycan synthesis was an important factor in the promotion of calcification.

Creatine kinase activity is required for mineral deposition and matrix synthesis in endochondral growth cartilage

In earlier studies, we have drawn attention to the unique changes in energy metabolism that accompany the maturation of epiphyseal growth plate chondrocytes. The objective of this investigation was to examine the importance of the ATP generating enzyme creatine kinase (CK), in the development and mineralization of the growth plate. We inhibited CK function by administering β-guanidinopropionic acid (β-GPA) to rats in vivo and to cultured chick chondrocytes in vitro. We found that this agent inhibited normal development of cartilage. Disorganization of chondrocytes in the proliferative and hypertrophic zones, poor vascular invasion, and retention of calcified cartilage occurred in the long bones of β-GPA-fed rats. β-GPA caused a change in the electrophoretic mobility of type II and type X collagens. Inhibition of apatite formation in the bones of shell-less chick embryos was accompanied by a CK isoenzyme shift from a bone-specific phenotype to a CK isozyme profile similar to that of cartilage. The results of these studies indicate that CK activity is required for normal development of the growth plate and that interference with creatine phosphate metabolism results in profound changes in the synthesis of cartilage and the maturational activities of chondrocytes.

Expression of immunoglobulin genes in the avian embryo bone marrow revealed by retroviral transformation.

Analysis of the early stages of avian B lymphocyte differentiation has been hampered by the low frequency of extra-bursal B lineage cells in sites of hematopoiesis. Consequently, little is known about B lineage precursors prior to their migration into the bursa of Fabricius. Colonization of the bursa typically occurs between about days 8 and 14 of embryonic (e) development, although cells which can colonize the bursa, functionally defined as pre-bursal stem cells, can be demonstrated in embryo bone marrow up until about the time of hatch. As a novel approach to analyzing early stages of avian B lymphocyte development, we show here that transformed B lineage cells can be derived from chick embryo bone marrow after infection in vitro with the replication-defective retrovirus REV-T produced in the context of the non-cytopathic CSV helper virus. Thus, exposure of day 14e-15e chick embryo bone marrow cells to REV-T (CSV) results in the generation of transformed, polyclonal lines of cells. From these lines, cells expressing cell surface immunoglobulin were readily isolated by flow cytometric cell sorting and single cell cloning. Analysis of the phenotype of REV-T(CSV)-transformed clones with a panel of monoclonal antibody reagents demonstrated that transformation by v-rel likely leads to marked changes in cell surface antigen expression. Nonetheless, clones expressing cell surface immunoglobulin expressed apparently normal mRNA for immunoglobulin mu and light chain and contained apparently normal immunoglobulin heavy and light chain gene rearrangements. Furthermore, no evidence for chromosomal deletions or aberrations of the Ig loci was detected among either sIg+ or sIg- REV-T(CSV)-transformed clones.

Expression of collagen type transcripts in chick embryonic bone detected by in situ cDNA-mRNA hybridization

The development of the chick embryonic calvarium, an intramembranous bone, is characterized by direct differentiation of cranial ectomesenchymal cells into osteoblasts without the formation of a cartilage anlage. Collagen biosynthesis remains predominantly as type I in the calvaria. However, in severely calcium-deficient chick embryos maintained in shell-less (SL) culture, cartilage-specific type II collagen is synthesized by the calvaria. Immunohistochemistry localized the cells expressing type II collagen to undermineralized regions of the SL bone. In this study, collagen gene expression in bones of normal (N) and calcium-deficient SL chick embryos was examined at Incubation Day 14 by in situ cDNA-mRNA hybridization. A critical step in the procedure, which used biotinylated cDNA probes, was the selection of fixation conditions which maximized RNA retention and maintenance of tissue morphology. Tissues fixed in modified Carnoy's fixative (58% ethanol, 30% choloroform, 10% acetic acid, 2% formaldehyde) for 2–4 hr at −20°C sectioned well and retained their cell morphology and cytoplasmic RNA. Other treatments important for the procedure included demineralization in 0.25 M HCl and removal of matrix by hyaluronidase digestion. In situ hybridization with type-specific collagen cDNA probes revealed that type II collagen mRNA was present in cells throughout the SL calvaria. More importantly, cells with type II collagen mRNA were also present in N calvaria which do not synthesize the protein. The overall abundance of type II-positive cells in N calvaria was not significantly different from that in SL calvaria, but their distribution throughout the bones differed. In general, the regional distribution of type II cells was inversely correlated with the extent of matrix mineralization. In the N calvaria, cells containing collagen type II mRNA were absent in the extensively mineralized superior zone, but were found in the temporal zone which showed limited mineralization. On the other hand, in the SL calvaria, which were substantially undermineralized overall, cells with type II mRNA were found throughout the tissue. Interestingly, the overall ratio of type I cells to type II cells was approximately 50% higher in N calvaria. These findings suggest that collagen type mRNA expression in the chick embryonic calvarium is correlated with, and perhaps dependent on, the extent of tissue matrix mineralization.

The effectiveness of inhibitors of soluble prolyl hydroxylase against the enzyme in the cisternae of isolated bone microsomes

Inhibitors of purified, soluble prolyl hydroxylase ( K. Majamaa et al. (1984) Eur. J. Biochem. 138, 239–245; K. Majamaa et al. (1986) J. Biol. Chem. 261, 7819–7823 ) were tested against isolated chick embryo bone microsomes containing intracisternal prolyl hydroxylase and its radiolabeled, unhydroxylated procollagen substrate. Two groups of inhibitors were used which consisted of pyridine-2-carboxylate and 1,2-dihydroxybenzene (catechol) derivatives. The 2,4- and 2,5-pyridine dicarboxylic acids, which are potent inhibitors of the soluble enzyme ( K i values 2 and 0.8 μ m, respectively), were effective in the same concentration range against intracisternal prolyl hydroxylase, although their relative affinities were reversed. Inhibition by pyridine-2,4-dicarboxylate in the microsomal system was reversed by increasing the concentration of 2-oxoglutarate. Pyridine-2,4-dicarboxylic acid did not inhibit the uptake of 2-[ 14C]oxoglutarate into microsomes, so it appears likely that the inhibitor must traverse the microsomal membrane and act directly at the enzyme level. Pyridine-2-carboxylic acid was ineffective in the microsomal system at 1 m m whereas it is a relatively potent inhibitor of the soluble enzyme with a K i of 25 μ m. This finding suggests that the second carboxyl group of the pyridine carboxylate derivatives may be required for their transport into the microsomal lumen. In the soluble system, 3,4-dihydroxybenzoic acid and 1,2-dihydroxybenzene had been found to be competitive inhibitors with relatively low K i values of 5 and 25 μ m, respectively. In the microsomal system, half-maximal inhibition was obtained at approximately 50–100 μ m and inhibition was not reversed by increasing the concentrations of either 2-oxoglutarate or ascorbate, alone or together. These results imply that in situ these compounds do not inhibit prolyl hydroxylase directly. Thus, the microsomal system can assess the accessibility of the intracisternal enzyme to potential inhibitors and offers an insight into the in cellulo potential of such compounds.

Chemical and biological characterization of low-molecular-weight human skeletal growth factor

Skeletal growth factor (SGF) activity was extracted from human bone matrix by demineralization and purified under dissociative conditions using hydroxyapatite, HPLC gel-filtration and HPLC reverse-phase chromatography. Human SGF thus purified was characterized chemically and biologically. Purified human SGF stimulated chick embryo bone cell proliferation at picomolar concentrations (half maximum at 2–3 ng/ml) and had little or no activity on other cell types tested (mouse 3T3 and normal rat kidney fibroblasts, embryonic chick intestinal and human placental cells). Human SGF did not displace 125I-labeled epidermal growth factor finding to normal rat kidney cells and did not stimulate normal rat kidney cell colony formation in soft agar. Human SGF activity was sensitive to trypsin, chymotrypsin, papain, dithiothreitol and performic acid but was resistant to heat (upto 70°C), pH (3–10), cyanogen bromide, alkaline phosphatase and neuraminidase and did not bind jack bean concanavalin A or kidney bean lectin. From our chemical and biological studies it appears that human SGF is different from other known polypeptide growth factors: epidermal growth factor, fibroblast growth factor, insulin, insulin-like growth factor-I, platelet-derived growth factor and transforming growth factor.

Human skeletal growth factor stimulates collagen synthesis and inhibits proliferation in a clonal osteoblast cell line (MC3T3-E1).

Human skeletal growth factor (hSGF), an 11-kD polypeptide purified from human bone, has been proposed to be a local regulator of bone formation. To investigate the underlying cellular mechanisms in an in vitro model system, we examined the effects of hSGF on proliferation and collagen synthesis in cells of the clonal osteoblast cell line MC3T3-E1. This line was isolated from newborn mouse calvarial cells and retains many characteristics of mature osteoblasts (Sudo, H., et al., (1984) J. Cell Biol. 96:191). A 14-hr treatment with hSGF increased noncollagenous protein synthesis to 215% of unstimulated controls and increased collagen synthesis to 630% of controls as determined by [3H]proline incorporation and high-pressure liquid chromatographic separation of [3H]proline and [3H]hydroxyproline in acid hydrolysates of trichloroacetic acid-insoluble protein. HSGF did not increase cell number over a 48-hr period and caused a reversible inhibition of DNA synthesis. Half-maximal hSGF concentration for stimulation of [3H]proline incorporation and inhibition of [3H]thymidine incorporation was 100 ng/ml. HSGF also inhibited DNA synthesis in cells stimulated by serum. In contrast, hSGF stimulated both collagen synthesis and DNA synthesis in primary cultures of chick embryo bone cells, which may be developmentally less mature than MC3T3-E1 cells. The results suggest that hSGF directly stimulated mature osteoblast matrix synthetic activity and that hSGF has differential effects on proliferation of osteoblast progenitor cells and mature osteoblasts.

Characterization of collagen hydroxylysyl glycosyltransferases as mainly intramembranous microsomal enzymes.

The localization of collagen hydroxylysine galactosyl- and galactosyl-hydroxylysine glucosyltransferases in purified chick embryo bone microsomes was studied by differential solubilization with nonionic detergents. Brij-35 (polyoxyethylene 25-lauryl ether) which selectively releases intracisternal proteins, and Triton X-100, whose specificity varies with its concentration, were used in the presence or absence of high ionic strength NaCl. These methods were used previously to characterize prolyl hydroxylase as intracisternal and lysyl hydroxylase as mainly intramembranous. The distribution of both glycosyltransferases within microsomes was similar to that of lysyl hydroxylase; approximately 70-80% of their activities are intramembranous with the remainder intracisternal. Collagen hydroxylysine glucosyltransferase differed from prolyl and lysyl hydroxylase and the galactosyltransferase in that its activity in vitro was apparently inhibited by membrane vesicles, even in the presence of detergents at concentrations which permeabilize the membrane. Accurate measurement of its activity could be achieved only by its separation from vesicles after detergent treatment. The common location of the major portion of lysyl hydroxylase and the glycosyltransferase activities suggests that they may act as a multienzyme complex to preferentially modify certain lysyl residues in nascent procollagen chains as they traverse the membrane of the endoplasmic reticulum. Since these enzymes do not act on helical collagen, their physical separation from prolyl hydroxylase may ensure that modifications of lysine residues occur prior to formation of hydroxyproline, which stabilizes the helical form.

Formation of proline metabolites in chick embryo bone: Interference with the measurement of free hydroxyproline by ion-exchange chromatography

Metabolites of l-[ 14C]proline were found in the trichloroacetic acid-soluble fraction of 16-day-old chick embryo frontal bones. In several ion-exchange procedures these metabolites interfered with the analysis of hydroxyproline derived from the metabolic breakdown of collagen. The major metabolite was identified as glutamic acid by its chromatographic and crystallization properties. It was eluted from AG50 cation-exchange resin with 1.0 n HCL in the hydroxyproline region, but was separated from hydroxyproline on a DC-6A column in the amino acid analyzer. Another metabolite was identified as aspartic acid. It was not separated from hydroxyproline on either AG50 using 1 n HCL for elution or on DC-6A using 0.1 m sodium citrate, pH 3.25, for elution, but adequate separation was obtained by elution with 0.2 m sodium citrate buffer at pH 2.91. Formation of these metabolites was not related either to protein synthesis or proline hydroxylation. Therefore, it is possible to analyze for hydroxyproline accurately by using a separate unhydroxylated sample to correct for the presence of the metabolites. The formation of glutamic acid suggested that proline oxidase activity might be present in bone tissue, but none was detected using a sensitive radioisotopic assay. Although the amount of radioactivity found in the metabolites was 36% of the amount of [ 14C]proline incorporated into protein, no radioactive glutamic or aspartic acid was present in protein hydrolyzates. This observation suggests that the metabolites did not enter the major amino acid pool used for protein synthesis.

An intramembranous reductant which participates in the proline hydroxylation reaction with intracisternal prolyl hydroxylase and unhydroxylated procollagen in isolated microsomes from L-929 cells

We previously have described a substance present in crude sonicates of L-929 cells which replaced ascorbate in vitro as a reductant for prolyl hydroxylase ( B. Peterkofsky, D. Kalwinksy and R. Assad, 1980, Arch. Biochem. Biophys. 199, 362–373). In the present study we found that almost 90% of the substance was particulate after differential centrifugation of stationary phase L-929 cell homogenates. The substance was not localized in nuclei or mitochondria and was found in the same fractions as microsomes, but these fractions also contained lysosomes and cell membranes. The reductant could not be solubilized from particles by Brij-35, indicating that it is an intrinsic component of a membrane rather than intracisternally located. The intramembranous cofactor, in the absence of ascorbate, participated in the in vitro hydroxylation of [4- 3H]proline in radio-actively labeled, intracisternal unhydroxylated procollagen in isolated microsomes which also contained prolyl hydroxylase. Hydroxylation was determined by measuring tritiated water formed from release of the 4- trans tritium atom. Since it is unlikely that such participation could occur if the cofactor were located within the membrane of another subcellular organelle, we have concluded that it is in the same particle as prolyl hydroxylase and unhydroxylated procollagen, that is, the microsome. With the endogenous reductant the reaction was slower than with saturating ascorbate and was increased by NADH. Maximum hydroxylation with the endogenous reductant was close to that which could be achieved with ascorbate. These results provide strong evidence that the endogenous reductant alone can account for the phenomenon of ascorbate-independent proline hydroxylation in L-929 cells. As in the case of ascorbate, the microsomal reductant functioned only in the presence of α-ketoglutarate and Fe 2+ and served as reductant for lysyl hydroxylase. It also was detected in the particulate fraction of virally transformed BALB 3T3 cells and in purified microsomes from bones of intact chick embryos. Since ascorbate could be taken up and concentrated in bone microsomes, it is unlikely that the endogenous reductant serves as an intermediary between ascorbate and intracisternal prolyl hydroxylase.

Regulation of collagen secretion by ascorbic acid in 3T3 and chick embryo fibroblasts

Ascorbate is required for optimal collagen secretion in Balb 3T3 fibroblasts and chick embryo bone fibroblasts. When 3T3 cultures are supplemented with ascorbate, there is a lag phase of approximately 45 min. before collagen secretion begins while in the absence of the vitamin, collagen accumulates in the cells until secretion begins at a slower rate after 1.5–2 hr. Collagen synthesized and secreted in the absence of ascorbate is extensively underhydroxylated (10–20% of optimum). In chick embryo cells, collagen secretion is also stimulated by ascorbate but, in addition, total collagen synthesis is decreased in the absence of the vitamin. It is suggested that the reduced secretion of collagen, which is probably caused by a deficiency of hydroxylation of proline and lysine in the absence of ascorbate, may eventually produce the defect in collagen synthesis which is observed in scorbutic animals.

Chick embryo development: chemical variations of cartilage.

AbstractThe chemical composition of cartilage from vertebrae and long bones of chick embryos 8 to 18 days old has been established. Collagen, muco‐polysaccharides, and non‐collagenous protein show similar patterns of distribution in the two types of cartilage; the maximum variation in these components has been found between the fourteenth and the sixteenth day of the prehatching period. The modifications that we have measured seem to be related more to a general biochemical feature of embryo development than to the preparation of the cartilage matrix for calcification.

Activation of Non Competent Embryonic Chicken Spleen and Bone Marrow Cells by Thymus and Bursa Fabricii Extracts

Previous studies have shown both thymus and bursa cells to activate a pre-existing competence in adult lymphoid cells, probably by means of a humoral factor, Klopstock and Sharon (1965), Miller (1965), Small and Trainin (1967).Rather than using intact cells as inducers we were interested in finding out whether an extract of thymus or bursa cells could be shown to activate immunologically immature cells.For this purpose, two sorts of inactive cells, Simonsen (1965), Burnet and Burnet (1960) were chosen:1. chick embryo bone marrow cells2. chick embryo lymphoid cellsTo measure the immunologic competence of these cells, following their expected activation by extract from bursa and/or thymus cells, we decided to use the Simonsen CAM phenomenon, Cock and Simonsen (1958). The Simonsen phenomenon, as is known, entails the production of focal lesions on the chorio-allantoic membrane (CAM) of chick embryos upon placement on the membrane of immunologically competent…

Stimulation by N6, O2′-dibutyryl adenosine 3′,5′-cyclic-phosphate of amino acid transport, protein synthesis, and chondroitin sulfate synthesis in embryonic bone in vitro, contrasted with the effects of thyroid hormones

Measurement were made of the effects of N 6,O 2′-dibutyryl adenosine 3′,5′-cyclic-phosphate on uptake and incorporation of amino acids and inorganic sulfate by intact pelvic bones of 10-day chick embryos. The N 6,O 2′-dibutyryl adenosine 3′,5′-cyclic-phosphate causes of 20–50% stimulation of transport of metabolizable and non-metabolizable neutral amino acids, of amino acid incorporation, and of inorganic sulfate incorporation. The effect of theophylline on amino acid transport was comparable to that of N 6,O 2′-dibutyryl adenosine 3′,5′-cyclic-phosphate but theophylline did not stimulate incorporation of amino acids or of sulfate. Cyclic 3′,5′-AMP (with or wihout theophylline), 5′-AMP, and potassium butyrate were without effect. Comparisons of the effects of N 6,O 2′-dibutyryl adenosine 3′,5′-cyclic-phosphate and of thyroid hormones on amino acid transport do not support the possibility that the stimulation by thyroid hormones is mediated by cyclic 3′-5′-AMP.

The distribution of calcium in normal and tetracycline-modified bones of developing chick embryos.

The presence of calcium (GBHA technique) in the periosteal cells, osteoblasts, osteocytes and chondrocytes of developing chick embryo bone confirms the findings ofKashiwa (1966) in the rat. Modification of bone development by tetracycline administration indicates that there is a direct correlation between the amount of calcium within these cells and the degree of mineralization of the adjacent matrices. The calcium of the calcification front of bone and the abnormally calcified cartilage matrix resulting from tetracycline treatment appears to differ from that of the older bone and normally calcifying cartilage matrix in its ability to chelate with GBHA. The distribution of the GBHA positive material appears to correspond to that of a pyridine-resistant bound lipid previously reported.

Preliminary Studies on Cell Cultures Infected with Marek's Disease Agent

Bioassay of Marek's disease (MD) agents presently requires chick inoculation and later examination for lesions (2,10,13). The usefulness of such methods is limited by the physical requirements of time, susceptible birds, and strict isolation facilities, and also by the subjectivity of the response measurement. The studies reported herein were based on the premise that the assay of MD agent in cell culture would constitute a significant improvement over existing in vivo methods. Previous attempts to propagate the MD agent in cell culture were either unsuccessful or equivocal. Biggs and Payne (2) failed to demonstrate infectivity in chick embryo fibroblast cultures inoculated with the B14 strain when tested at the 3rd and 6th passages. Vindel (11) propagated in cell culture a filtrable agent isolated from chickens with neurolymphomatosis. This agent produced a rapid cytopathic effect in chick embryo fibroblasts, and 30C of chickens inoculated with fluids from infected cultures developed MD within 3 months. The identity of this agent was not definitely established, however, since lesions occurring in the inoculated chicks were not described, and evidence that the lesions were induced by the inoculum was inconclusive. Despite the paucity of evidence for the propagation of MD agent in cell culture, the potential usefulness of a cell culture bioassay appeared to justify further investigation. The initial objective of this study was to develop techniques for cultivating MDinfected cells and for infecting cultures with MD agent. This report describes successful maintenance of the JM strain of MD in cultures of chick embryo bone marrow and certain other cells. We are aware of simultaneous and independent investigations by Calnek (4) and Kottaridis and Luginbuhl (7), as yet unpublished,