The effects of spaceflight on mammary metabolism in pregnant rats.

The effects of spaceflight on mammary metabolism of 10 pregnant rats was measured on Day 20 of pregnancy and after parturition. Rats were flown on the space shuttle from Day 11 through Day 20 of pregnancy. After their return to earth, glucose oxidation to carbon dioxide increased 43% (P < 0.05), and incorporation into fatty acids increased 300% (P < 0.005) compared to controls. It is unclear whether the enhanced glucose use is due to spaceflight or a response to landing. Casein mRNA and gross histology were not altered at Day 20 of pregnancy. Six rats gave birth (on Day 22 to 23 of pregnancy) and mammary metabolic activity was measured immediately postpartum. The earlier effects of spaceflight were no longer apparent. There was also no difference in expression of beta-casein mRNA. It is clear from these studies that spaceflight does not impair the normal development of the mammary gland, its ability to use glucose, nor the ability to express mRNA for a major milk protein.

Ectodermal stimulation of the production of hyaluronan-dependent pericellular matrix by embryonic limb mesodermal cells.

Interaction of ectoderm and underlying mesoderm is essential for normal vertebrate limb morphogenesis. One of the functions of limb bud ectoderm is its influence on the composition of extracellular matrix in subectodermal mesoderm, which in turn participates in morphogenesis of this region of the limb. This matrix is highly enriched in hyaluronan, even at the time when the level of hyaluronan in the chondrogenic and myogenic regions of the limb decreases, due to secretion of a stimulatory factor by the ectoderm. In this study we show that limb bud ectoderm not only stimulates hyaluronan synthesis but induces formation of large hyaluronan-dependent, pericellular matrices around cultured limb bud mesodermal cells. The ectodermal activity is mimicked in great part by fibroblast growth factor-2 and transforming growth factor-beta, and antibodies to these proteins inhibit induction of mesodermal pericellular matrix by the ectodermal factor. It has been shown by other investigators that fibroblast growth factor-2 is produced by limb ectoderm whereas transforming growth factor-2 is produced by limb ectoderm whereas transforming growth factor-beta is present in limb mesodermal tissues. Thus we conclude that the unique properties of mesodermally produced matrix underlying limb bud ectoderm are regulated, at least in part, by ectodermal fibroblast growth factor-2, probably in concert with mesodermal transforming growth factor-beta.

Hyaluronan-dependent pericellular coats of chick embryo limb mesodermal cells: induction by basic fibroblast growth factor.

Basic fibroblast growth factor (FGF) has been shown previously to be present in the chick embryonic limb during early stages of its development, at which time the limb mesodermal cells are proliferating within a hyaluronan-rich extracellular matrix. In this study, basic FGF was found to stimulate hyaluronan synthesis and production of hyaluronan-dependent pericellular coats by mesodermal cells from the chick embryo limb; acidic FGF, platelet-derived growth factor, epidermal growth factor, and retinoic acid either had a much smaller effect than basic FGF or an inhibitory effect. Transforming growth factor-beta stimulated hyaluronan synthesis and coat formation but, unlike basic FGF, this factor also stimulated chondroitin sulfate production by the mesodermal cells.

Hyaluronate-cell interactions and growth factor regulation of hyaluronate synthesis during limb development.

Hyaluronate is a major component of the intercellular matrix surrounding proliferating and migrating cells in embryonic tissues. When placed in culture, mesodermal cells from the early, proliferative stages of limb development produce high levels of hyaluronate and exhibit prominent hyaluronate-dependent pericellular coats. Cells from the subsequent stages of mesodermal condensation that precede differentiation to cartilage and muscle produce less hyaluronate and do not exhibit these coats. Also at this time, binding sites specific for hyaluronate appear on the surface of the mesodermal cells. These binding sites may participate in the mechanism of condensation by mediating cell aggregation and the endocytosis of hyaluronate. Further changes in hyaluronate-cell interaction occur during differentiation of the condensed mesoderm to cartilage and muscle. Hyaluronate synthesis and pericellular coat formation in the mesoderm are stimulated by a factor, related to transforming growth factor-beta, that is produced by the surrounding ectoderm. The early limb also contains high levels of basic fibroblast growth factor. Its concentration is highest at the earliest stages, when cell proliferation and hyaluronate synthesis are prominent activities, and this factor has been shown to stimulate both these activities in cultures of limb mesodermal cells. Thus fibroblast growth factor and transforming growth factor-beta may be important in the regulation of early growth and morphogenesis of the limb.

Developmental changes in fibroblast growth factor in the chicken embryo limb bud.

Cell proliferation is a major event during early limb development. Significant levels of growth factor activity, as measured by stimulation of DNA synthesis in mouse BALB/c 3T3 cells, were found in extracts of chicken embryo limb buds at early stages of development. Extracts from stage-18 limbs (3 days of incubation) were 2 to 3 times more potent than were extracts from older stages, namely 22-24 (4 days), 26 (5 days), and 28 (6 days). Basic fibroblast growth factor (bFGF) was measured specifically using an RIA, and the amounts of factor obtained corresponded to the activities measured by the 3T3 cell-growth assay. In addition, most growth factor in the extracts bound with high affinity to heparin-Sepharose columns. Western (immunologic) blotting and immunoprecipitation with an antibody specific for bFGF revealed a protein of identical size to bFGF--i.e., 18 kDa, in the extracts. Thus, a growth factor with the properties of bFGF is present in the early limb, and the level of this factor is highest when proliferation is a predominant cellular event in the developing limb. These and other data suggest that fibroblast growth factor is a key regulatory factor in embryonic growth and morphogenesis.

Transferrin and the growth-promoting effect of nerves.

In addition to its role in the activity of specialized proteins such as hemoglobin and myoglobin, iron is required as a cofactor in several important enzymes common to most animal cells. One such enzyme, ribonucleotide reductase, which regulates the production of deoxyribonucleotides during DNA synthesis, requires a continuous supply of iron to maintain its activity throughout the process of DNA replication. The mechanism by which animal cells normally acquire iron involves receptor-mediated uptake of iron-loaded transferrin, followed by release of apotransferrin. The density of transferrin receptors on the cell surface is greatly increased in rapidly dividing normal and neoplastic cells. Various mitogens and certain organogenic tissue interactions have been shown to induce the appearance of transferrin receptors, signalling the onset of DNA replication. Interference with this process of iron delivery causes the rapid arrest of cell cycling, frequently during the S phase itself, which underscores the importance of iron for DNA replication. Although most circulating transferrin is synthesized in the liver and embryonic yolk sac, smaller quantities are produced in several other embryonic organs and certain other adult tissues. It has been suggested that local synthesis and/or release of transferrin supplies the iron required by rapidly growing cells in situations where the cells do not have ready access to adequate amounts of plasma transferrin due to incomplete development of the vasculature or the presence of blood-tissue barriers (Ekblom and Thesleff, 1985; Meek and Adamson, 1985). Oligodendrocytes and Schwann cells have been shown to synthesize and/or contain high concentrations of transferrin and these cells therefore may constitute a local source of this factor for neurons, whose growth and survival in vitro require transferrin. Transferrin in central and peripheral nervous tissues may be significant for the trophic or growth-promoting effect neurons exert on cells of certain tissues. Transferrin duplicates the activity of neural tissue or neural extracts on growth and development of cultured skeletal myoblasts from chick embryos and on proliferation of mesenchymal cells in blastemas from regenerating amphibian limbs, two systems that have been widely used in investigations of the growth-promoting influence of nerves. Moreover, removal of active transferrin from neural extracts, either with antibodies to transferrin or chelation of the iron, inhibits reversibly the effect of the extract in these developing systems. While the physiological significance of the extract in these developing systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Transferrin and the trophic effect of neural tissue on amphibian limb regeneration blastemas.

Nerves promote regeneration of amputated urodele limbs, but the chemical basis of the effect is not known. We have examined the possible involvement of the iron-transport factor transferrin, which is important for cell proliferation and is present in vertebrate nervous tissue. Newt brain extract stimulated incorporation of [3H]thymidine in cultured blastemas from regenerating newt forelimbs, showing a biphasic dose-response similar to that of heterologous transferrin. As shown previously for transferrin, the inhibitory effect of brain extract at high concentrations was relieved by the addition of iron. Activity of brain extract was reduced by treatment with an iron-chelating agent and fully restored by the readdition of iron. Double immunodiffusion of newt tissue extracts and antibodies against newt plasma transferrin demonstrated the presence of transferrin-like factors in brain, spinal cord, and peripheral nerve. These results indicate that activity of transferrin may be part of the trophic effect of brain extract on cultured blastemas.

Changes in the extracellular matrix and glycosaminoglycan synthesis during the initiation of regeneration in adult newt forelimbs.

The extracellular matrix (ECM) of the distal tissues in a newt limb stump is completely reorganized in the 2-3-week period following amputation. In view of numerous in vitro studies showing that extracellular material influences cellular migration and proliferation, it is likely that the changes in the limb's ECM are important activities in the process leading to regeneration of such limbs. Using biochemical, autoradiographic, and histochemical techniques we studied temporal and spatial differences in the synthesis of glycosaminoglycans (GAGs) during the early, nerve-dependent phase of limb regeneration. Hyaluronic acid synthesis began with the onset of tissue dedifferentiation, became maximal within 1 weeks, and continued throughout the period of active cell proliferation. Chondroitin sulfate synthesis began somewhat later, increased steadily, and reached very high levels during chondrogenesis. During the first 10 days after amputation, distributions of sulfated and nonsulfated GAGs were both uniform throughout dedifferentiating tissues, except for a heavier localization near the bone. Since nerves are necessary to promote the regenerative process, we examined the neural influence on synthesis and accumulation of extracellular GAGs. Denervation decreased GAG production in all parts of the limb stump by approximately 50%. Newt dorsal root ganglia and brain-derived fibroblast growth factor each produced twofold stimulation of GAG synthesis in cultured 7-day regenerates. The latter effect was primarily on synthesis of hyaluronic acid. The results indicate that the trophic action of nerves on amphibian limb regeneration includes a positive influence on synthesis and extracellular accumulation of GAGs. Since the ECM exerts a major influence on cellular proliferation and migration, the effect of nerves on GAG metabolism may have considerable importance for growth and development of the early regenerate.

"Trophic" effect of transferrin on amphibian limb regeneration blastemas.

In light of the recent demonstration that one "neurotrophic factor" of peripheral nerves is the iron-transport glycoprotein transferrin, we tested the effects of heterologous transferrin on cellular events in cultured newt forelimb blastemas. Addition of transferrin to medium containing 1% fetal bovine serum resulted in DNA labeling and mitotic activity approximately twice as high as that of blastemas cultured in medium with 1% serum alone. Blastemas maintained for 24 hr in medium with 1% serum were stimulated to increased levels of DNA synthesis by the addition of transferrin, and this response was dose-dependent. Varying the concentrations of iron and transferrin in the medium gave results indicating that the glycoprotein's trophic effect is due to its ability to furnish iron to the cells in an appropriate manner. Results of the study are consistent with the hypothesis that blastema cell proliferation is promoted by transferrin or transferrin-like factors released from nerves.