Isolation, characterization and synthesis of alpha-foetoprotein from neonatal-rat skin.

Monospecific anti-[rat alpha-foetoprotein(alpha-FP)] immunoglobulin G was coupled to CNBr-activated Sepharose-4B (4.5 mg/ml packed volume of gel) to yield an adsorbent. The immunoaffinity column was used to isolate alpha-FP from neonatal-rat skin. Purified skin alpha-FP was found to be immunologically and electrophoretically similar to serum alpha-FP. It yielded a single band with mol.wt. 68000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, on polyacrylamide-gel electrophoresis under non-denaturing conditions, the alpha-FP displayed slow- and fast-moving variants similar to those observed in serum alpha-FP. A Scatchard plot of oestradiol binding to the alpha-FP yielded an association constant of 2.5 X 10(9)M-1 by dextran-coated-charcoal and 0.75 X 10(8)M-1 by Sephadex-gel-filtration procedures respectively. Skin explants from newborn rats were found to incorporate [14C]leucine into immunoprecipitable intracellular alpha-FP. Cycloheximide inhibited the synthesis of alpha-FP in skin explant culture. Our results indicate that newborn-rat skin contains alpha-FP that is similar to serum alpha-FP and which may arise in neonatal-rat skin as a result of synthesis in situ.     

Induction of lipid synthesis in mammary organ cultures from mature virgin and pregnant goats.

The hormones needed to induce lipogenesis in mammary organ cultures from mature virgin and pregnant goats were studied. In tissues from both mature virgin goats and goats at week 10 of pregnancy, cultivated in Waymouth medium without hormones, the rate of the incorporation of (1-(14C))-acetate into the lipids was low and decreased throughout culture. In the presence of insulin, the rate of acetate incorporation was maintained at a higher level. Cortisol acted synergistically with insulin, to produce a rate of lipid synthesis higher than that using insulin alone. The further addition of prolactin had little effect on the incorporation of acetate into the lipids of mammary explants from mature virgin goats, but markedly stimulated it in tissue from animals at weeks 9--10 of pregnancy. The maximum increase in the rate of lipid synthesis was achieved in the presence of 0.5 microgram prolactin/ml, whereas with growth hormone 50 microgram/ml was needed for the maximum effect. The initial rate of acetate incorporation into mammary explants from goats at weeks 13 and 18 of pregnancy was high. It was not stimulated by the hormones during culture, however, and decreased more rapidly in the absence of hormones than in their presence. The rate of acetate incorporation into the lipids was in agreement with the histological evaluation of the secretory response of the mammary explants after cultivation. The secretory response to prolactin and the rate of the incorporation of acetate into the lipids were highest in goats at weeks 9--10 of pregnancy while in tissues from goats at weeks 13 and 18 were not stimulated and decreased during culture.     

Human bronchial explants in long-term culture: Establishing a baseline for secretion

Summary A serum-free culture medium was employed to evaluate both qualitative and quantitative aspects of secreted proteins derived from human bronchial explants over a period of 26 to 50 d. It was found that protein and hexose were secreted at a reasonably constant rate during this period. Viability of explants was assessed by histological examination, attachment to scored grids of the petri dish, incorporation of labeled precursors into tissue proteins, and by fluorographic analysis of the sodium dodecyl sulfate-polyacrylamide gel pattern of the secreted material. This culture system is useful as a model for the study of secretory products and processes and how they are affected by various stimuli.     

Wound closure in foetal rat skin.

Foetal rat skin rapidly closes an open wound in organ culture and in vivo, this possibly being unique to organs still in the morphogenetic stage. In the present study, examination was made of morphological changes in foetal rat skin during closure of open wounds inflicted at day 16 of gestation. Phase-contrast microscopy of open-wounded skin cultured in vitro indicated inward spreading of the peripheral skin to be responsible for wound closure. Wound closure in vitro was inhibited by cytochalasin B (10 micrograms/ml), not by hydroxyurea (2 mM), indicating prenatal wound closure to be mediated by regulation of the microfilament system rather than cell proliferation. During wound closure in vitro and in vivo, light and scanning electron microscopy of the peripheral skin showed cells in the periderm, the outermost layer of the foetal epidermis, to elongate centripetally and en masse, whereas the shape of underlying epidermal cells not to change. Numerous spindle-shaped cells and fibrous matrices in the mesenchyme were redistributed, becoming oriented along the wound edge. Following isolation of the mesenchyme and epidermis by treatment with Dispase and separate culturing, the capacity for wound closure in vitro was found to be retained only by the mesenchyme. Cellular activity within the mesenchyme, rather than in the epidermis, would thus appear essential to wound closure in foetal rat.     

Effect of maternal ethanol consumption on foetal and neonatal rat hepatic protein synthesis.

Effects of maternal ethanol consumption were investigated on the rates of protein synthehsis by livers of foetal and neonatal rats both in vivo and in vitro, and on the activities of enzymes involved in protein synthesis and degradation. The rates of general protein synthesis by ribosomes in vitro studied by measuring the incorporation of [14C]leucine into ribosomal protein showed that maternal ethanol consumption resulted in an inhibition of the rates of protein synthesis by both foetal and neonatal livers from the ethanol-fed group. The rates of incorporation of intravenously injected [14C]leucine into hepatic proteins were also significantly lower in the foetal, neonatal and adult livers from the ethanol-fed group. Incubation of adult-rat liver slices with ethanol resulted in an inhibition of the incorporation of [14C]leucine into hepatic proteins; however, this effect was not observed in the foetal liver slices. This effect of externally added ethanol was at least partially prevented by the addition of pyrazole to the adult liver slices. Pyrazole addition to foetal liver slices was without significant effect on the rates of protein synthesis. Cross-mixing experiments showed that the capacity of both hepatic ribosomes and pH5 enzyme fractions to synthesize proteins was decreased in the foetal liver from the ethanol-fed group. Maternal ethanol consumption resulted in a decrease in hepatic total RNA content, RNA/DNA ratio and ribosomal protein content in the foetal liver. Foetal hepatic DNA content was not significantly affected. Ethanol consumption resulted in a significant decrease in proteolytic activity and the activity of tryptophan oxygenase in the foetal, neonatal and adult livers. It is possible that the mechanisms of inhibition of protein synthesis observed here in the foetal liver after maternal ethanol consumption may be responsible for at least some of the changes observed in 'foetal alcohol syndrome'.     

Precocious development of uridine diphosphate glucuronosyltransferase activity during organ culture of foetal rat liver in the presence of glucocorticoids.

1. Precocious development of mammalian UDP-glucuronosyltransferase (EC 2.4.1.1.7) induced by endogenous compounds of known chemical composition is reported for the first time. 2. This development occurs in cultured explants of foetal rat liver when exposed to corticosteroids possessing a pregn-4'-ene structure and a hydroxy or an oxo group at C-11. 3. Explants from 14-day foetuses cultured for 3 days in a chemically defined medium containing dexamethasone exhibited transferase activities towards o-aminophenol within adult male values. Those liver transferase activities attained in utero by 17 days were still negligible. 4. Evidence from several approaches indicated that the explants required glucocorticoids for expression of the transferase, not for maintenance of viability. 5. Glucocorticoid-dependent stimulation of transferase activity required incorporation of L-[14C]leucine into protein, as judged from the pulsing of cultures with cycloheximide. 6. The relevance of these culture experiments to the situation in vivo is discussed.     

Synthesis and secretion of albumin and transferrin by foetal rat hepatocyte cultures

Hepatocytes derived from foetal rat liver synthesize and secrete albumin and transferrin when maintained in primary culture. These proteins are produced for at least seven days under the conditions of culture. Studies on hepatocyte cultures derived from 12, 13, 14, 15 and 19-day foetal rats show that the maximal cellular rate of secretion of both proteins increases about 50-fold over this period. The maximal rate of albumin secretion in all cultures is achieved after one day in culture and decreases in hepatocytes from early foetuses after the fourth to sixth day in culture. Transferrin secretion by hepatocytes from 12 to 15 day foetuses increases markedly during the second day of culture and is relatively constant thereafter. In contrast, secretion of transferrin by hepatocytes from 19-day foetuses is constant from the first day of culture. The results show that both albumin and transferrin are synthesized and secreted by the foetal liver as early as the twelfth day of gestation. The increase in the rate of transferrin secretion that occurs during culture of hepatocytes from 12 to 15 day foetuses may reflect the development of a secretory mechanism that is different from that for albumin.     

Influence of torpor on milk protein composition and secretion in lactating bats.

In the pipistrelle bat (Pipistrellus pipistrellus), the metabolic load of lactation is not met to any significant extent by increased food intake or mobilization of body reserves, and aerial foraging accounts for most of the animal's energy expenditure even during lactation. Energy conservation must, therefore, play a critical role in maintaining lactation. The principal mechanism for energy conservation appears to be the bat's ability to enter torpor, but this may itself interrupt milk synthesis and secretion unless the pipistrelle mammary gland is adapted to counteract its effect. The effect of torpor on mammary tissue function was studied in mammary tissue explant cultures prepared in weeks 1-3 of lactation, when milk water yield was 0.20, 0.88, and 0.30 mL/d respectively. Protein synthesis measured by incorporation of radiolabeled amino acids was 44% lower (P < 0.001) in bat tissue explants cultured at ambient temperature (22 degrees C) compared with 37 degrees C. The reduction was similar to that observed in mouse mammary tissue (57%) and was unaffected by stage of lactation. Analysis of explant protein after [35S]methionine labelling showed the majority of proteins synthesised in culture to be milk proteins; it also demonstrated that the decrease in protein synthesis at ambient temperature was a general phenomenon: synthesis of both secretory and intracellular mammary proteins was reduced at the lower culture temperature. The results suggest that bat mammary tissue has no mechanism to counteract the effect of reduced body temperature and that periods of lactational torpor are likely to cause a pronounced diurnal variation in the rate of milk secretion.     

Casein turnover in rabbit mammary explants in organ culture

1. Explants of mammary gland from mid-pregnant rabbits were cultured in medium 199 containing insulin, prolactin and cortisol, and specific anti-casein immunoglobulin G was used to measure the amount, rate of synthesis and rate of degradation of casein in the explants in the presence of hormones and after removal of hormones from previously stimulated tissue. 2. The amount of casein in particle-free supernatants prepared from mammary explants was measured by ;rocket' immunoelectrophoresis. 3. The rate of incorporation of l-[4,5-(3)H]leucine into casein was measured after isolation of the casein by immunoadsorbent chromatography and polyacrylamide-gel electrophoresis in the presence of urea and sodium dodecyl sulphate. 4. Casein accumulates in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in a decrease in the rate of accumulation of casein in the explants. 5. Casein-synthetic rate increases in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in continued casein synthesis at approx. 30% of the rate in the presence of hormones. The synthetic rate does not decrease to values observed in explants cultured throughout in the absence of hormones. 6. Casein is not degraded in mammary explants during a phase of rapid casein accumulation (36-72h) in the presence of hormones. Furthermore casein is not degraded when hormones are removed from the tissue after between 36 and 72h in culture. 7. Casein is glycosylated in mammary explants; the extent of glycosylation parallels the rate of synthesis. The glycosylated protein is rapidly secreted from the tissue. 8. The results are consistent with the notion that after hormonal stimulation mammary explants from mid-pregnant rabbits synthesize, glycosylate and rapidly secrete casein. Removal of hormones decreases the synthetic rate of casein, but does not cause the accumulation of a pool of degradable casein in the lobuloalveolar cells.     

eIF2alpha phosphorylation, stress perception, and the shutdown of global protein synthesis in cultured CHO cells

The perception of environmental stress in animal cells engineered to produce heterologous protein leads to the induction of stress signaling pathways and ultimately apoptosis and cell death. Protein synthesis is regulated in response to various environmental stresses by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2). In this study we have utilized a model system of Chinese hamster ovary cells engineered to secrete recombinant TIMP-1 protein to investigate the relationship between the cellular rate of protein synthesis, eIF2alpha phosphorylation, cellular stress perception, and the rate of cell specific recombinant protein synthesis. The rate of total protein synthesis was maximal after 48 hours of culture, remaining relatively high until 96 hours of culture, after which a decline was observed. Towards the end of culture a marked increase in labeled secreted protein was observed. Total eIF2alpha expression levels were high during the exponential growth phase and decreased slightly towards the end of culture. On the other hand, the relative expression of phosphorylated eIF2alpha showed a bi-phasic response with a small increase in phosphorylated eIF2alpha observed at 48 hours of culture, and a significant increase at 120 hours post-inoculation. The large increase in phosphorylated eIF2alpha coincided with the observed increase in labeled secreted protein and the decline in total cellular protein synthesis. A marked increase in ubiquitination was also observed at 120 hours post-inoculation that coincided with reduced rates of cellular protein synthesis and mRNA translation attenuation. We suggest that eIF2alpha phosphorylation is an indicator of cellular stress perception, which could be exploited in recombinant protein manufacturing to commence feeding and engineering strategies.     

Hormonal control of casein synthesis in mammary explants from pregnant goats

The effects of insulin, cortisol, prolactin, 3,3',5-triiodo-L-thyronine (L-T3) and progesterone on the synthesis of total protein and casein in mammary explants from pregnant goats were studied. In the absence of hormones and in the presence of insulin plus cortisol the rate of incorporation of 14C-leucine into proteins that were precipitated with the anti-casein antibody decreased during culture. The addition of prolactin to hormonal combination of insulin and cortisol caused large stimulation of rates of casein synthesis. Maximum incorporation of leucine was attained between 3 and 5 days of culture in the presence of 0.5 microgram ml-1 of prolactin. Prolactin stimulated-casein and total protein synthesis were not consistently affected by the addition of L-T3 or progesterone. The inhibition of DNA synthesis by hydroxyurea or cytosine-arabinofuranoside had no effect on casein synthesis in mammary explants from pregnant goats.     

Origin of corticosteroid-binding globulin in fetal rat. Comparative dynamics of corticosteroid-binding globulin, alpha-fetoprotein, and albumin secretion in primary cultures of fetal rat hepatocytes

The origin of corticosteroid-binding globulin (CBG) and its evolution in comparison with alpha-fetoprotein (AFP) and albumin synthesis, during early development of rat liver (days 13 and 15 of fetal life), have been investigated using cultured fetal hepatocytes. Synthesis and secretion of CBG, AFP, and albumin is evidence by cycloheximide-sensitive [14C]leucine incorporation into immunoprecipitable polypeptides secreted by cultured hepatocytes into the medium, two-dimensional immunoelectrophoretic and autoradiographic identification of newly synthesized labeled proteins, corticosterone and estradiol-17 beta binding to CBG and AFP, respectively, and indirect immunofluorescence localization of AFP, albumin, and CBG in cultured fetal hepatocytes. CBG, albumin, and AFP accounted for 6, 11, and 25% (in 13-day-old rat fetuses) and 5, 15, and 28% (15-day-old rat fetuses), respectively, of the total secreted proteins in the culture medium. The rates of CBG, AFP, and albumin (counts/minute of secretion [14C]leucine incorporated per milligram of cell protein/hour of culture) in the hepatocytes of 15-day-old rat fetuses were 1.48-, 2.1-, and 2.57-fold higher, respectively, than in the 13-day-old rat fetuses. These results indicate that fetal liver is also active in CBG synthesis, along with AFP and albumin, as early as day 13 of fetal life and that the synthetic rates of these secretory proteins depend upon the developmental stage of the fetal liver. This developmental related change in the rate of synthesis of CBG by the fetal hepatocytes may regulate the level of free (active) glucocorticoid in the fetal circulation and thereby the initiation and regulation of glucocorticoid-dependent processes during the crucial stages of the differentiation of fetal liver and other developing tissues.     

Induction of surfactant protein in fetal lung. Effects of cAMP and dexamethasone on SAP-35 RNA and synthesis

Synthesis of surfactant-associated glycoprotein of Mr = 30,000-35,000 (SAP-35) was induced in explant culture of human fetal lung obtained from 8 to 24 weeks of gestation. SAP-35 synthesis and content increased markedly during 1-5 days in organ culture in association with the morphologic maturation of Type II epithelial cells and the appearance of lamellar bodies. [35S] Methionine labeling of the explants and subsequent immunoprecipitation of SAP-35 demonstrated distinct high-mannose precursors and sialylated SAP-35 forms as early in culture as SAP-35 synthesis was detectable. The increase in SAP-35 synthesis was associated with increased SAP-35 RNA of 2.1 kilobases as assessed by hybridization assay with [32P]cDNA specific for human SAP-35. Specific SAP-35 RNA increased during organ culture and both SAP-35 content and SAP-35 RNA increased in the absence of exogenous hormones in 2% carbon-stripped fetal calf serum. SAP-35 content and synthesis was stimulated by 8-Br-cAMP. Addition of 100 microM 8-Br-cAMP, enhanced both the concentration of SAP-35 protein and the SAP-35 RNA as assessed by hybridization assay. In contrast, treatment of the explants with dexamethasone was associated with decreased SAP-35 protein synthesis, SAP-35 content, and decreased SAP-35 RNA levels compared to untreated explants. Inhibition by dexamethasone occurred at all gestational ages tested, was dose-dependent, and detectable within 24-48 h during organ culture. Dexamethasone significantly inhibited both basal and cAMP-induced SAP-35 synthesis. Induction of pulmonary surfactant protein (SAP-35) synthesis during organ culture of human fetal lung was associated with increased SAP-35 RNA. SAP-35 synthesis and SAP-35 RNA were inhibited by dexamethasone and enhanced cAMP.     

Regulation of enzyme turnover during tissue differentiation. Interactions of insulin, prolactin and cortisol in controlling the turnover of fatty acid synthetase in rabbit mammary gland in organ culture

1. Explants of mammary gland from mid-pregnant rabbits were cultured in Medium 199 containing combinations of insulin, prolactin and cortisol. With hormone combinations which included prolactin, a sustained increase in the apparent rate of synthesis and in the amount of fatty acid synthetase was measurable immunologically. Maximum increase was produced with insulin, prolactin and cortisol present together. 2. With prolactin present alone, synthetase activity in the explants decreased to undetectable values after 1 day in culture, whereas the incorporation of l-[U-(14)C]leucine into immunodetectable material increased. Prolactin may therefore direct the synthesis of immunologically cross-reactive precursors of fatty acid synthetase which are enzymically inactive. 3. Culture with dibutyryl cyclic AMP plus theophylline in the presence of insulin, prolactin and cortisol delayed the increase in the rate of synthesis and accumulation of the synthetase. These compounds may also prevent the apparent decrease in the rate of degradation of the synthetase which occurs on day 2 of culture. 4. A large decrease in the apparent rate of degradation of the synthetase on day 2 of culture occurs during culture with hormone combinations which include prolactin. The protein obtained by centrifugation of explant homogenates for 6min at 14000g(av.) is degraded continuously throughout the culture period. 5. This decrease in the apparent rate of degradation of the synthetase was measured by radio-immunological precipitation. It is probably part of a regulated programme of enzyme degradation and not a reflexion of the reutilization of radioactive amino acids for the following reasons. (a) The calculated increase in the amount of the synthetase in explants on day 2 of culture with insulin, prolactin and cortisol was approximately equal to the measured increase of the enzyme complex which accumulates in the explants. This suggests little or no enzyme degradation has occurred. (b) Explants were cultured for 24h with insulin, prolactin and cortisol. They were then incubated with l-[U-(14)C]leucine, washed and incubated again for up to 4(1/2)h. l-[U-(14)C]Leucine rapidly equilibrated with the intracellular amino acid pool. Within 10min of incubation after washing explants to remove endogenous l-[U-(14)C]leucine the previously linear incorporation of l-[U-(14)C]-leucine into total explant protein ceased. This suggests that protein is synthesized from an amino acid pool which rapidly equilibrates with amino acids in the culture medium. (c) Explants were cultured for 24h as described in (b) but after washing they were cultured with insulin, prolactin and cortisol for 24h. Approx. 90% of the radioactivity lost from the ;free' intracellular amino acid pool and from amino acids derived from the degradation of explant protein in this period was detected in the culture medium. This suggests that the ;free' intracellular amino acids and amino acids derived from protein degradation can equilibrate with amino acids in the medium. A residual ;free' radioactive amino acid pool was present in the tissue. (d) Casein represents approx. 20% of the protein synthesized after 1 day in culture with insulin, prolactin and cortisol. Histological evidence suggests that on day 2 of culture, casein is unlikely to be degraded in the tissue. No increase in the radioactivity incorporated into casein can be measured in the 23h after incubation of explants with l-[U-(14)C]leucine as described in (b). This suggests that the incorporation of radioactivity into proteins during culture after incubation with l-[U-(14)C]leucine is minimal. (e) Inhibition of protein synthesis in explants by cycloheximide after incubation with l-[U-(14)C]leucine does not reveal a latent continuous degradation of fatty acid synthetase on day 2 of culture which might have been masked by the high rates of protein synthesis and therefore the accumulation of the enzyme. 6. The conclusion is discussed that there is a real decrease (or even cessation) in the rate of degradation of fatty acid synthetase during the period when the enzyme accumulates in explants cultured with hormone combinations which contain prolactin.     

Mechanisms of stimulation of collagen synthesis during chick embryonic skin development.

To study how collagen synthesis is regulated in developing chick embryonic skin, hydroxyproline synthesis, incorporation of proline, and translational activity and content of collagen mRNA in 12-, 15-, and 18-day skins were determined and compared with each other. Hydroxyproline synthesis in the 18-day skins was markedly increased over that in the 12-day skins, whereas proline incorporation was moderately increased. The increase in collagen synthesis from day 15 to 18 was accompanied by increases in both the translational activity and the content of type I procollagen mRNA, with a selective increase in the lower-molecular-weight species of pro alpha 1 (I) collagen mRNA. In contrast, the stimulation of collagen synthesis from day 12 to day 15 did not parallel the levels of type I procollagen mRNA. These results suggest that the stimulation of collagen synthesis is regulated by collagen mRNA levels only in the later stage of development (from day 15 to day 18). Both the collagen synthesis and type I procollagen mRNA levels in the fibroblasts isolated on each corresponding day were constant. The difference in collagen synthesis under two different culture conditions suggests that cell-matrix interaction and/or some serum factors, including several growth factors, are essential for the marked stimulation of collagen synthesis observed in 12- to 18-day skin.     

Human skin fibroblast collagenase inhibitor. Comparative studies in human connective tissues, serum, and amniotic fluid

In order to gain insight into the biological significance of a collagenase inhibitor secreted by human skin fibroblasts, we examined various human connective tissues and body fluids for such a protein. The inhibitors found in these tissues were compared immunologically to skin fibroblast inhibitor by Ouchterlony analysis and by the development of a highly specific enzyme-linked immunosorbent assay (ELISA). Using this ELISA, cell cultures of human skin fibroblasts, corneal fibroblasts, gingival fibroblasts, and adult and fetal lung fibroblasts secreted similar amounts of immunoreactive inhibitor protein. Each culture medium displayed a reaction of immunologic identity with skin fibroblast inhibitor when examined in Ouchterlony gel diffusion. In testing for functional inhibitory activity, the same 1:1 stoichiometry of collagenase inhibition was observed in each culture medium that characterizes the human skin inhibitor. Other mesodermally derived human cell types, including human fetal osteoblasts, uterine smooth muscle cells, fibrosarcoma cells, and explants of tendon and articular cartilage behaved in the same manner as the fibroblast cultures. Because collagenase inhibitors with biochemical similarities to skin fibroblast inhibitor have been described in serum and in amniotic fluid, we also examined these sources of inhibitory proteins. The data indicate that both serum and amniotic fluid contain collagenase inhibitors which are immunologically and functionally identical with the skin fibroblast inhibitor. The concentration of inhibitor in serum, as measured by ELISA assay, is 1.03 +/- 0.27 micrograms/ml. The results suggest that collagenase inhibitors which are functionally equivalent and immunologically identical with human skin fibroblast collagenase inhibitor are synthesized by many, if not all, fetal and adult mesodermal tissues in the human organism. The inhibitor apparently gains access to certain body fluids such as serum and amniotic fluid. This inhibitor protein may, therefore, function in the regulation of collagen degradation in most human connective tissues.     

Hormonal interactions in mammalian collagenase regulation: Comparative studies in human skin and rat uterus

The production of collagenase by human skin explants in culture is prevented by 10^?8 M dexamethasone, 5·10^?4 M dibutyryl cyclic AMP, or 2.5· 10^?3 M theophylline. Decreases in collagenase activity are paralleled by reductions in the degradation of explant collagen during the culture period. Progesterone, which effectively inhibits collagenase production in rat uterine explant cultures, has no effect on human skin explants. The inhibition by cyclic AMP is nucleotide specific. When partially inhibitory concentrations of dexamethasone and dibutyryl cyclic AMP, or dexamethasone and theophylline, are added to culture medium together, the resultant inhibition is that predicted by additivity. Synergistic inhibition, as observed in rat uterus between progesterone and dibutyryl cyclic AMP, fails to occur.Dexamethasone inhibits the production of collagenase by cultured explants of rat uterus, with complete inhibition occurring at 10^?7 M steroid. Synergism between glucocorticoids and dibutyryl cyclic AMP or between dexamethasone and progesterone could not be demonstrated in the uterine culture system. These results suggest the existence of three regulatory systems for the control of collagenase production in mammalian tissues, and that cooperativity between systems may occur on a tissue-specific basis.     

Metallothionein synthesis in foetal, neonatal and maternal rat liver

The synthesis of hepatic metallothionein relative to other cytosol proteins was measured by [35S]cysteine incorporation in foetal, neonatal and pregnant rats. The relative rate of hepatic metallothionein synthesis reached a maximum in foetal liver on days 18-21 of gestation. Metallothionein synthesis then declined until weaning, when adult levels were established. The rate of metallothionein synthesis was greater in pregnant rats at term than in nulliparous rats. To determine if circulating inducing agents could play a role in the regulation of metallothionein synthesis in foetal liver we treated pregnant rats with inducers at a time prior to the normal rise in foetal liver metallothionein synthesis. Injections of copper, cadmium or hydrocortisone to 17-day-pregnant dams failed to induce foetal metallothionein synthesis. In contrast, zinc injection to the dam was an effective inducer in the foetuses. Maternal laparotomy (performed to expose the foetus for direct injection of inducers) induced foetal metallothionein synthesis. Metallothionein synthesis in the livers of 17-day-gestation dams was induced by all metal injections and laparotomy but, surprisingly, not by hydrocortisone injection. Maternal adrenalectomy did not influence the subsequent normal elevation in foetal or maternal metallothionein synthesis. These results, in conjunction with previous reports, suggest that mobilization of zinc in serum during late gestation may regulate foetal and maternal changes in metallothionein synthesis.     

Organ culture of adult rat colonic mucosa on fibrin foam

A system for maintaining adult rat colonic mucosa in organ culture for up to 28 days is described. Distal colonic mucosa physically separated from the muscle layers was cultured at 37 degrees C on a substrate of human fibrin foam in HEPES- and bicarbonate-buffered Waymouth's MB 752/1 medium supplemented with 10% fetal bovine serum, L-glutamine, bovine albumin, ascorbic acid, hydrocortisone, insulin, and ferrous sulfate; the optimal atmostphere for culture was 95% O2 and 5% CO2. Viability of explants was demonstrated by tissue morphology with light microscopy, incorporation of [3H]thymidine and [3H]leucine into DNA and protein, [14C]glucosamine and [3H]fucose incorporation, and glycoprotein synthesis. Two days after initiation of culture, degeneration of surface and crypt cells was observed. Secreted mucosubstances covered the explants. Explants maintained in 95% O2 retained a variable number of glandular crypts with normal columnar epithelium for 14 to 21 days in culture. At 28 days, explants contained a single layer of cuboidal surface epithelium and a rare cryptlike gland.     

Increased epsilon(gamma-glutamyl)lysine crosslinking associated with increased protein synthesis in the inner layers of healing rat skin wounds.

Three days after biopsy wounds were made in the dorsal skin of rats the animals were killed and explants of wounded and unwounded skin were incubated for 7 h with either [3H]glutamine or [3H]lysine. Both incubated and fresh control explants were then dissected into three layers which were homogenized, extracted, digested and then assayed for epsilon (gamma-glutamyl)lysine. The concentration of epsilon(gamma-glutamyl)lysine was greater in all three wounded layers than in the corresponding unwounded layers. The concentration in the wounded middle (dermal) layer and in the unwounded middle layer of younger rats was greater than in the unwounded outer (keratinized) layer, which has previously been shown to contain epsilon(gamma-glutamyl)lysine crosslinks. The incorporation of label from both [3H]glutamine and [3H]lysine into buffer-insoluble protein of the middle and inner (muscle) layers was much greater in the wounded explants than in the unwounded. Except for [3H]lysine in the inner layer there was also an increase in the fraction of incorporated label which was converted to epsilon(gamma-glutamyl)lysine. These results show that increased protein biosynthesis during repair in the wounded explants is associated with increased formation of epsilon(gamma-glutamyl)lysine. In addition, they indicate that the crosslink is involved in some process in the middle and inner layers which is distinct from its known function in keratinization of the epidermis.