Secretion-coupled protein degradation: studies on mammary casein

Mammary explants from midpregnant rabbits were cultured for 18 h at 37 degrees C with insulin, prolactin and cortisol. Subsequently, explants were labelled for 2 h with inorganic [32P]phosphate, L-[5-3H]proline or L-[4,5-3H]leucine, washed and chased for up to 3 h. The radiolabelling profile of [32P]casein or [3H]casein during the chase period, obtained by isoelectric focussing or immunoprecipitation indicates extensive destruction of neosynthesized casein. The extent of casein destruction in mammary explants in culture (measured after radiolabelling with L-[5-3H]proline), is inversely related to casein secretion. Least casein degradation is observed in explants after 48 h in culture when casein secretion is maximal (observed histochemically). Subsequently, when the extracellular alveolar lumen is filled with secretion products (72 h), rapid intracellular casein destruction is again observed. When the chase was carried out in the presence of drugs which inhibit degradation and/or secretion, the results indicate that secretion-coupled casein degradation is dependent on an intact functional microfilamentous-microtubular network, casein is not degraded by an autophagosome requiring process, degradation is inhibited by leupeptin, amino-acid analogue containing casein does not undergo secretion-coupled degradation and inhibition of N-glycosylation of intracellular vesicular membrane proteins prevents secretion-coupled degradation. Secretion-coupled protein destruction is discussed in relation to the post-translational regulation of the net production of secretory proteins in eukaryotic cells.     

Regulation of intracellular casein degradation by secreted milk proteins

Intracellular degradation of newly synthesised casein was measured by a pulse-chase method in freshly prepared goat mammary explants. After incubation in medium containing L-[5-3H]proline, explants were washed and cultured again in unlabelled medium containing 5 mM proline; at intervals up to 24 h the amount of radiolabel incorporated in casein was measured. Tissue was obtained in week 33 of lactation after goats had been milked incompletely in one gland (the test gland) for 24 weeks; the contra-lateral (control) gland was milked normally. In explants from the control gland, casein was not degraded during or after secretion: L-[5-3H]proline incorporated in casein increased to a maximum value which was maintained through the chase period. For four out of five goats, explants from the test gland showed a decrease in total [3H]casein radiolabel at 0-4 h of the chase, indicating that a proportion of casein was degraded during secretion. Intracellular casein degradation was also observed when control gland explants were cultured in chase medium containing a goat whey fraction known to inhibit casein production and milk secretion (Wilde, C.J. et al., (1987) Biochem. J. 242, 285-288). This suggests that the greater volume of residual milk left by incomplete milking reduced secretory efficiency, rendering casein susceptible to intracellular degradation, and that this occurred through the action of a secreted milk constituent, which acts as a chemical feedback inhibitor 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.     

Possible interaction of cyclic nucleotides with the prolactin stimulation of casein synthesis in mouse mammary gland explants.

During a 10-h incubation, cyclic nucleotide phosphodiesterase inhibitors, viz. theophylline and quinine, were found to reduce by 40-50% the rate of [3H] leucine incorporation into casein in mammary gland explants from midpregnant mice. Further, dibutyryl cyclic AMP as well as the phosphodiesterase inhibitors were found to abolish the prolactin stimulation of leucine incorporation into casein. Elevated levels of cyclic AMP therefore appear to impair the functionality of the mammary gland. Although cyclic GMP was previously shown to stimulate RNA synthesis in the mammary gland in a prolactin-like manner, it had no effect on the rate of casein synthesis in mammary gland explants. Preincubation of explants with cyclic GMP did, however, attenuate the time required for the commencement of the prolactin stimulation of the rate of leucine incorporation into casein. A physiological role of cyclic GMP for the regulation of the rate of casein synthesis is thus suggested.     

The effect of calcium on synthesis and degradation of mammary cytosolic proteins and casein

In mammary explants prepared from mid-pregnant rabbits, rates of intracellular and secretory protein synthesis and degradation can be manipulated by the addition or removal of hormones (insulin, prolactin, cortisol). Under both culture conditions, depletion of extracellular calcium with EGTA markedly decreases cytosolic protein synthesis and degradation, and also decreases the rate of synthesis of casein. Culture of explants in calcium-free medium also inhibits cytosolic protein synthesis, but has no significant effect on protein degradation, either in the presence or the absence of hormones. The results suggest that mammary protein synthesis and degradation may be affected by changes in the intracellular concentration or distribution of calcium.     

Interleukin-1 receptor antagonist delivered directly and by gene therapy inhibits matrix degradation in the intact degenerate human intervertebral disc: an <Emphasis Type="Italic">in situ</Emphasis> zymographic and gene therapy study

Data implicate IL-1 in the altered matrix biology that characterizes human intervertebral disc (IVD) degeneration. In the current study we investigated the enzymic mechanism by which IL-1 induces matrix degradation in degeneration of the human IVD, and whether the IL-1 inhibitor IL-1 receptor antagonist (IL-1Ra) will inhibit degradation. A combination of in situ zymography (ISZ) and immunohistochemistry was used to examine the effects of IL-1 and IL-1Ra on matrix degradation and metal-dependent protease (MDP) expression in explants of non-degenerate and degenerate human IVDs. ISZ employed three substrates (gelatin, collagen, casein) and different challenges (IL-1β, IL-1Ra and enzyme inhibitors). Immunohistochemistry was undertaken for MDPs. In addition, IL-1Ra was introduced into degenerate IVD explants using genetically engineered constructs. The novel findings from this study are: IL-1Ra delivered directly onto explants of degenerate IVDs eliminates matrix degradation as assessed by multi-substrate ISZ; there is a direct relationship between matrix degradation assessed by ISZ and MDP expression defined by immunohistochemistry; single injections of IVD cells engineered to over-express IL-1Ra significantly inhibit MDP expression for two weeks. Our findings show that IL-1 is a key cytokine driving matrix degradation in the degenerate IVD. Furthermore, IL-1Ra delivered directly or by gene therapy inhibits IVD matrix degradation. IL-1Ra could be used therapeutically to inhibit degeneration of the IVD.     

Protein degradation during terminal cytodifferentiation. Studies on mammary gland in organ culture.

1. In mammary gland explants subjected to experimental manipulation, average rates (during 24 h periods) of degradation of fatty acid synthase, casein and cytosol-fraction proteins were measured by a double-isotope method. Rates of degradation of fatty acid synthase were also computed from measurements of changing enzyme amount and rate of synthesis. 2. During the period of most rapid enzyme accumulation there is a transient decrease in the computed rate of degradation of fatty acid synthase. Removal of hormones produces a rapid increase in the computed rate of degradation of the enzyme. 3. The average rate of degradation of fatty acid synthase measured by the double-isotope method is low in the presence of hormones, and increases on hormone removal. This increase in degradation rate is inhibited by adrenaline and further blocked by insulin. NH4Cl (10 mM) also partially inhibits the increase in protein degradation on hormone removal. 4. The pattern of changes in the average rate of degradation of cytosol-fraction proteins is similar to that for fatty acid synthase alone. There is no relationship between subunit molecular weight and rate of degradation under all experimental conditions. 5. Isotope ratios for resolved cytosol protein mixtures are transformed logarithmically to make the standard deviations an estimate of heterogeneity of degradation rates. By this analysis, in some conditions there appears to be significant measureable heterogeneity of degradation rates. 6. Little degradation of casein is measured in the presence of hormones, but a marked increase in the rate of degradation can be measured when hormones are removed. Whereas at 24-48h NH4Cl (10 mM) has little effect on this enhanced rate of degradation, at 48-72h it causes a large decrease in degradation rate. 7. Results are discussed in terms of a two-component degradation system in mammary gland explants.     

The differential actions of cortisol on the synthesis and turnover of alpha-lactalbumin and casein and on accumulation of their mRNA in mouse mammary gland in organ culture.

Cortisol was previously shown to exert different, concentration-dependent, effects on the accumulation of casein and alpha-lactalbumin in mammary glands from mid-pregnant mice cultured in the presence of insulin and prolactin [Ono & Oka (1980) Cell 19, 473-480]. The present study demonstrated that the addition of 30nM-cortisol to the medium containing insulin and prolactin resulted in a marked enhancement of the rate of synthesis of both alpha-lactalbumin and casein in cultured tissue. The addition of 3 microM-cortisol in combination with insulin and prolactin caused a marked decrease in the rate of alpha-lactalbumin synthesis, but increased casein synthesis substantially. Similar changes were also observed in the amount of translatable mRNA for alpha-lactalbumin and casein in mammary explants cultured with insulin, prolactin and the two concentrations of cortisol. The study of the turnover of the milk proteins in cultured explants showed that virtually all of the casein synthesized remained intact in tissue explants cultured with 3 microM cortisol, whereas about 45% of casein disappeared in 40h from explants cultured with 30nM-cortisol. In contrast, the two concentrations of cortisol did not differentially affect the disappearance of alpha-lactalbumin, which was about 55% in 40h. These results indicate that the concentration-dependent differential actions of cortisol on the accumulation of alpha-lactalbumin and casein are exerted through its effects on the rate of synthesis and turnover of the two proteins as well as on the accumulation of their mRNA species.     

Functional differentiation in mouse mammary gland epithelium is attained through DNA synthesis,inconsequent of mitosis

Virgin mouse mammary gland in explant culture will differentiate and synthesize casein and α-lactalbumin when insulin, hydrocortisone, and prolactin (IFPRL) are present in the culture medium. Explants whose DNA synthesis has been blocked differentiate cytologically, mobilize lipid, synthesize RNA, and incorporate ³H-amino acids into proteins to the same extent as unblocked tissue. Nevertheless, casein synthesis as measured by immunoprecipitation with casein-specific antiserum remains at the zero-time level in blocked explants while unblocked explants produce casein at five- to eightfold greater levels. Electrophoretic analysis of immunoprecipitated radioactive proteins showed that the IFPRL-treated virgin tissue made all four size classes of mouse casein. Immunoperoxidase studies of explants revealed that the number of mammary epithelial cells positive for casein was 2–8% in blocked and 24–31% in unblocked, in good agreement with the radioimmunoprecipitation results. Immunoelectron microscopy demonstrated the accumulation of casein within the cisternae of the granular endoplasmic reticulum and in Golgi vacuoles in the unblocked epithelial cells. Similar accumulation did not occur in blocked cultures despite the secretory appearance of the cells. Autoradiographic analysis of blocked and unblocked explants, incubated in the presence of IFPRL and [³H]thymidine for 72 hr, showed that 53–57% of the epithelial cells synthesized DNA in unblocked explants, whereas only 2% incorporated the label in the presence of cytosine arabinoside. When explants were incubated with IFPRL and various concentrations of colchicine, only 5–6% of the epithelial cells were found to enter mitosis. Since cell duplication cannot account for the severalfold increase in casein-producing cells in the unblocked explants, the results suggest that the requirement for DNA synthesis in this system may involve either polyploid cells or the augmentation of specific sequences necessary for the facilitation of terminal differentiation. Similar requirements for DNA synthesis were not observed in mammary explants from pregnant and primiparous (but nonpregnant) mice.     

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.     

Type IV phosphodiesterase activity specifically regulates cAMP-stimulated casein secretion in the rat mammary gland

This study investigates the regulation of cAMP-stimulated casein secretion in rat mammary explants by cAMP phosphodiesterase (cAMP-PDE) activity. cAMP-PDE activity of the lactating rat mammary gland is shown to be provided by three families, types II, III and IV. In mammary explants, general inhibition of the cAMP-PDE activity significantly increased the rate of cAMP-stimulated casein secretion. This effect could be mimicked using the type-IV specific inhibitor rolipram but not by the specific, or combined, inhibition of the type II and type III activity. Only type IV activity significantly affected intracellular accumulation of cAMP whereas all three cAMP-PDE activities were shown to influence the PKA activation ratio in cells. RtPCR analysis showed that the mammary gland apparently expresses just three type IV isozymes, RNPDE4A5, RNPDE4A8 and RNPDE4D3. A specific role for type IV cAMP-PDE activity in the regulation of casein secretion is suggested and possible mechanisms for the effects of PDEIV activity discussed.     

An enzyme related to the high molecular weight multicatalytic proteinase, macropain, participates in a ubiquitin-mediated, ATP-stimulated proteolytic pathway in soluble extracts of BHK 21/C13 fibroblasts

Soluble, cell-free extracts of BHK 21/C13 fibroblasts degraded a variety of exogenous proteins to acid-soluble peptides at pH 8.0. ATP stimulated the rates of proteolysis. Both the absolute rate of proteolysis and the magnitude of the ATP effect depended on the specific substrate. For example, casein was degraded approximately 10-fold faster than lysozyme, but lysozyme degradation was more highly stimulated by ATP than was casein degradation. Ubiquitin enhanced the ATP-stimulated proteolysis of each substrate in both postmicrosomal extracts and DEAE-cellulose fractionated extracts. In each extract, ubiquitin enhanced the ATP-stimulated degradation of lysozyme to a greater degree than that of casein. These results suggested that lysozyme was degraded by a pathway that was more dependent upon ubiquitin than was casein. Further evidence for this conclusion was obtained in studies using substrates whose amino groups were blocked by extensive methylation or carbamoylation. The high molecular weight proteinase, macropain, appears to be involved in the ATP-stimulated degradation of both substrates. Specific immunoprecipitation of macropain with polyclonal antibodies resulted in the inhibition of ATP-stimulated proteinase activity both in the absence and presence of ubiquitin. These results indicate that macropain plays a role in both ubiquitin-mediated and ubiquitin-independent ATP-stimulated proteolysis in BHK cell extracts.     

Rapid suppression of protein degradation in skeletal muscle after oral feeding of leucine in rats

A diet containing adequate amounts of protein rapidly suppresses myofibrillar protein degradation in rats and mice. This study determined whether dietary amino acids inhibit postprandial protein degradation in rat skeletal muscle. When rats fed on a 20% casein diet for 1 h after 18 h starvation, the rate of myofibrillar protein degradation measured by N(tau)-methylhistidine release from the isolated extensor digitorum longus muscle was significantly (p < 0.05) decreased at 4 h after refeeding. A diet containing an amino acid mixture which is the same composition as casein also reduced myofibrillar protein degradation at 4 h after refeeding (p < 0.05). An essential amino acid mixture (15.1%, corresponding to casein composition) and a leucine (2.9%) diets reduced the rate of myofibrillar protein degradation after refeeding (p < 0.05), whereas a protein free diet did not. Administration of leucine alone (0.135 g/100 g body weight) by a feeding tube induced a decrease in the rate of myofibrillar protein degradation at 2 h after administration (p < 0.05), whereas the serum insulin concentration was constant after leucine administration. These results suggested that leucine is one of regulating factors of myofibrillar protein degradation after refeeding of a protein diet.     

Cortisol induces rapid accumulation of whey acid protein mRNA but not of asl and b-casein mRNA in rabbit mammary explants

Mammary explants from rabbit were cultured in the presence of various combinations of insulin, cortisol and prolactin. The concentration of whey acidic protein (WAP) asl-casein and b-casein mRNA was measured using specific cDNA probes. Medium alone was added to the explants for one day. Prolactin with and without cortisol was then added to the medium. Prolactin alone induced rapidly asl-and b-casein gene but not WAP gene. When cortisol was added with prolactin, the asl- and b-casein genes were induced at the same rate as in the absence of the steroid. In contrast, the WAP mRNA was then rapidly accumulated. This induction process was not altered by cycloheximide for two hours and it was blocked at a later stage. In a second experiment, insulin and prolactin were first added for 24 hours in the culture medium. Cortisol was then added and the concentration of the three mRNA was measured. Cortisol did not significantly modify the level of asl- and b-casein mRNA. On the contrary, the WAP mRNA was rapidly accumulated. These data indicate that the well-established amplificatory effect of glucocorticoids on casein gene expression is a slow process whereas their effect on the WAP gene is rapid. This suggests that glucocorticoids induce casein gene expression through an indirect cellular mechanism not involving a glucocorticoid receptor element in casein gene promoters and that WAP gene is more classically stimulated through the direct binding of the steroid receptor to a glucocorticoid receptor element located in its promoter.     

Lactogenic response of mouse mammary explants from different days of pregnancy to placental lactogen and pituitary prolactin

The lactogenic response of mouse mammary gland explants to human placental lactogen (hPL) and ovine pituitary prolactin (oPRL) was examined on days 10 to 18 of pregnancy by measuring 3H-amino acid incorporation into calcium-rennin precipitable casein. To determine the lactogenic response of the explants, the mean slopes of dose-response curves were calculated for each hormone treatment. Slope means of dose-response curves for oPRL and hPL did not differ from each other on any day of pregnancy examined. A triphasic pattern of response was suggested when slope means of dose-response curves for both hormones were plotted as a function of day of gestation. Peak responses were observed on days 10, 13 and 17-18. Combinations of oPRL and hPL, in ratios of oPRL:hPL = 2:1 and oPRL:hPL = 1:2, also produced a triphasic pattern of sensitivity very similar to that produced by either hormone alone. These results suggest that mouse mammary explants may be more sensitive to oPRL and hPL on days 10, 13 and 17-18 of pregnancy.     

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.     

Collagen degradation in the rabbit skin during short-term tissue culture

Full thickness rabbit skin explants were cultured on plastic dish for 1 week and the sequential morphological changes were examined daily by light and electron microscopy. During the cultured period, bundles of dermal collagen fibres gradually loosened and were removed from the upper dermis and from the cut margin of the explant, which was covered by a sheet of migrating epidermal cells. In these areas, cells containing phagocytosed collagen fibrils were observed from the 3rd day to the end of the culture period. These cells containing phagocytosed collagen fibrils included dermal fibroblasts and macrophages, epidermal keratinocytes and endothelial cells lining blood vessels. The presence of acid phosphatase activity in vacuoles containing the collagen fibrils suggested that intracellular degradation of collagen was occurring. In addition, extracellular collagen degradation was recognized around fibroblasts and beneath the migrating epidermis by the high collagenolytic activity at these sites. These findings suggest that both intra- and extracellular collagen degradation may participate in collagen removal from dermal connective tissue in cultured skin explants.     

Rapid multiplication of Sapium sebiferum Roxb. by tissue culture

Multiple shoots formation and elongation was induced from stem explants of Sapium seedlings on media containing cytokinins. Leaf explants produced callus on a medium containing cytokinins, auxin, casein hydrolysate and coconut milk, which could be induced to form multiple shoots on transfer to a medium lacking casein hydrolysate, coconut milk and auxin. Rooting of isolated shoots by treatment with an auxin mixture (indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid) and transfer of the plantlets to field have also been successful.     

SYNTHESIS OF LACTOFERRIN AND CASEIN BY EXPLANTS OF BOVINE MAMMARY TISSUE

Synthesis of lactoferrin and casein by the bovine mammary gland was determined in an experimental model where lactation was maintained in one mammary half, while involution was induced in the contralateral half. Culture of explants with prolactin had no consistent effect on synthesis of casein or lactoferrin in tissue from either mammary half. Endotoxin and tumor necrosis factor-α generally decreased synthesis of casein and lactoferrin, suggesting that these inflammatory mediators are not directly responsible for increasing lactoferrin synthesis during mammary inflammation or involution. Synthesis of lactoferrin was increased and casein decreased in the involuting mammary half vs. the lactating half. These results suggest that local factors in the mammary gland play a role in the regulation of lactoferrin synthesis during involution.