A corticotropin releasing hormone receptor antagonist does not delay parturition in rats.

OBJECTIVE: To determine the effects of CP-154, 526, a corticotropin releasing hormone (CRH) receptor antagonist, on the length of normal rat gestation. STUDY DESIGN: Twenty-four timed-pregnant Sprague-Dawley rats were purchased for this study. The drug and placebo were administered to the animals using an osmotic pump surgically inserted in the dorsal subcutaneous space. Six animals received 6 mg/kg/day of the drug, six animals received 12 mg/kg/day of the drug and twelve animals received the placebo. The gestational period, weight of each pup and number of pups in each litter were recorded and compared in the drug group versus placebo group. RESULTS: No difference was noted in the gestational period of the drug and placebo rats. The mean weight of pups in both the drug and placebo groups was 6.18 g. The number of pups per litter were similar in the drug and placebo groups. CONCLUSION: Antagonism of CRH receptors in rats has no effect on the length of gestational period, pup weight or number of pups per litter. Further studies are needed to define the role of CRH and its antagonism in primate pregnancy, as has been done in sheep.

S-nitrosohemoglobin in the fetal circulation may represent a cycle for blood pressure regulation.

It has recently been demonstrated, in rats, that hemoglobin transports nitric oxide (NO), as S-nitrosocysteine, from the lungs to the peripheral tissues. This cycle may be involved in the regulation of blood pressure and efficient delivery of oxygen in adult animals. We sought to determine whether this model was applicable to the human fetus. Umbilical cord blood was obtained from deliveries between 37 and 42 weeks of gestation (n = 19). NO, released from erythrocyte s-nitrosohemoglobin (SNO-Hb), was determined by the Saville reaction and total plasma NO was determined by the Greiss reaction. SNO-Hb levels were found to be higher in the umbilical vein, [SNO]/[Hb] = 2.19 +/- 1.22 (X10(-3)), than in the artery, [SNO]/[Hb] = 1.45 +/- 0.66 (X10(-3)) (P < 0.001, Wilcoxon Signed Rank test). This supports the hypothesis that fetal blood pressure may be regulated by erythrocytes acting via a hemoglobin-based mechanism.

Hypoxia stimulates ecNOS mRNA expression by differentiated human trophoblasts.

Cytotrophoblasts isolated from normal human placenta cultured under normoxic conditions (20% O2, pO2 = 130 mmHg) for 48-72 h differentiate to a form which expresses high levels of hCG and which morphologically resembles syncytiotrophoblast. We had previously shown that hypoxia (0-1% O2, pO2 = 12-14 mmHg) blocks this differentiation process, although trophoblasts exposed to hypoxia for up to 96 h were completely viable. In this article we showed that trophoblast responds to hypoxia by expressing the hypoxia-sensitive DNA binding protein HIF-1. We also showed that in trophoblast cultured under normoxic conditions, expression of endothelial cell nitric oxide synthase (ecNOS) mRNA increases with time, reaching a maximum in 48-72 h. However, in trophoblast maintained under hypoxic conditions for 48 h (after an initial 24 h in normoxia), expression of ecNOS mRNA is greatly reduced. These observations are consistent with the expression of ecNOS by syncytiotrophoblast but not by cytotrophoblast. In contrast, exposure of differentiated trophoblasts to hypoxia for 24 h (after 48-72 h in normoxia) significantly stimulates expression of ecNoS mRNA over that of cells maintained continuously in normoxia. These results suggest that in differentiated trophoblast hypoxia can stimulate ecNOS expression.

The effect of hypoxia on human trophoblast in culture: morphology, glucose transport and metabolism.

The response to hypoxia of trophoblast isolated from term placenta and maintained in culture was studied. Trophoblast exposed to normoxic (PO2 120-130 mmHg) or hypoxic (PO2 12-14 mmHg) conditions were examined by electron microscopy. After 48 h, the cytoplasm of the hypoxic cells was more electron-dense with increased numbers of mitochondria, lysosomes and vacuoles. Compared to normoxic cells, the surface microvilli of the hypoxic cells were sparse, short and unevenly distributed. [3H]thymidine incorporation by both hypoxic and normoxic trophoblast fell rapidly and equivalently after 2 days in culture. The percentage of cells with the proliferation-associated nuclear antigen, Ki 67, also decreased, but remained higher in hypoxic cells suggesting that hypoxia retarded completion of the cell cycle (normoxia, 10.80 +/- 2.51 s.e.; hypoxia, 19.87 +/- 2.73, P < 0.01). Glucose consumption was elevated in hypoxia (3.73 +/- 1.07 s.e. mumol/10(6) cells/24 h) as compared to normoxia (1.46 +/- 0.83, P = 0.01). Although lactate production was consistently higher in hypoxia, the difference was not statistically significant (hypoxia 5.38 +/- 1.54 mumol/10(6) cells/24 h versus normoxia, 1.52 +/- 0.29, P = 0.07). After 48 h, uptake of [3H]2-deoxglucose ([3H]2DG) by hypoxic cells was reduced to 12 per cent +/- 4.3 s.e. of that in normoxic cells; return to normoxia resulted in recovery within 10 min. Lineweaver-Burk plots of [3H]2DG uptake indicated high affinity (KM 2.2 +/- 0.4 x 10(-4) M) and low affinity transporters (KM 4.5 +/- 1.6 x 10(-3) M). Northern blot analysis identified mRNA for GLUT1 and GLUT3. In hypoxia, steady-state GLUT1 and GLUT3 mRNA were approximately three- and 10-fold higher than in normoxia respectively. Inhibitors of oxidative metabolism of glucose increased the uptake of [3H]2DG within 2 h, whereas hypoxia reduced uptake. Hence, trophoblast in culture survives in extreme hypoxia, but manifests striking changes in morphology and in glucose metabolism and transport. Completion of cell cycle appears to be retarded.

Alpha 1-antitrypsin blocks the release of transforming growth factor-alpha from MCF-7 human breast cancer cells.

Human breast cancer cells synthesize and release a variety of growth-modulating substances in response to estrogen stimulation, and it is generally accepted that the growth-promoting effects of estrogens are due at least in part to this autocrine/paracrine mechanism. Several of these growth-modulating substances, including transforming growth factor-alpha (TGF alpha) and its analogs, have been shown to require pericellular proteolysis for activation or release. Recently, we reported that MCF-7 human breast cancer cells are able to synthesize alpha 1-antitrypsin (alpha 1-AT), the major elastase inhibitor in human serum, and that there is a negative correlation between anchorage-independent growth of MCF-7 cells in soft agar and synthesis of alpha 1-AT. The studies we present here were undertaken to gain an understanding of the mechanisms responsible for this observation. We show that release of TGF alpha from its membrane-bound precursor on MCF-7 cells is blocked by alpha 1-AT whether the cells were maintained in the presence or absence of estradiol and that there is a clear dose-response relationship between the alpha 1-AT concentration and both the release of TGF alpha and growth in soft agar. Consistent with this, TGF alpha release was increased in the presence of antibody to alpha 1-AT. In contrast, TGF alpha release and growth in soft agar were not blocked by peptide inhibitors specific for trypsin- or chymotrypsin-like enzymes. The alpha 1-AT concentration required for a half-maximal effect is lower for inhibition of TGF alpha release than it is for inhibition of colony formation (0.4 vs. 1.5 mumol/L). However, both values are in the range of concentrations one might expect at the cell surface in vivo. A new MCF-7 cell subline producing 10-fold higher levels of alpha 1-AT than its parent cell line was constructed by stable transfection of MCF-7 ML cells (a subline producing low levels of alpha 1-AT) with an alpha 1-AT complementary DNA. Growth in soft agar and release of TGF alpha were significantly decreased in cells transfected with the alpha 1-AT complementary DNA compared to those in cells transfected with vector alone, although, TGF alpha expression was the same. The above observations support a model for growth regulation in human breast ductal epithelial cells in which growth factor activation and release are dependent on the coordinate action of proteases and protease inhibitors. This model would predict that alpha 1-AT can act as a tumor suppressor in inhibiting the growth of breast cancer cells.

The effect of hypoxia on term trophoblast: hormone synthesis and release.

Isolated trophoblast in culture remained viable when exposed to severe hypoxia (Po2 12-14 mmHg) for at least 72 h as indicated by trypan blue exclusion and the synthesis and secretion of metabolically labelled proteins. However, release of hCG, hPL, progesterone and estradiol was reduced to < 10 per cent when compared to trophoblast in normoxia (Po2 120-130 mmHg). hCG mRNA was also reduced demonstrating interruption of synthesis at transcription. Acute exposure to hypoxia (2 h) suppressed progesterone release but not hCG, whereas inhibitors of oxidative phosphorylation suppressed hCG release but not progesterone. hCG release increases progressively during culture in normoxia, peaking at 72 h. Exposure of trophoblast to hypoxia for 48 h after 24, 48 and 72 h in normoxia interrupted this progression but did not suppress hCG release. Progesterone release, in contrast, was reduced by hypoxia. Exogenous dibutyryl cAMP increased hCG and progesterone release by normoxic trophoblast but not by hypoxic cells. Trophoblast returned to normoxia after 24 h in hypoxia increased hCG and progesterone release, suggesting early recovery. Conservation of oxygen and ATP by reducing hormone synthesis may contribute to survival of trophoblast in hypoxia.

Isolation, characterization, and properties of a trypsin-chymotrypsin inhibitor from amaranth seeds.

A trypsin-chymotrypsin inhibitor was isolated from the seeds of amaranth--a highly nutritious protein source. The purification of the inhibitor (AmI) was carried out by affinity chromatography on trypsin-Sepharose and by HPLC. AmI is a single-chain protein of 8 kD, as determined by electrophoresis on SDS-polyacrylamide gels and by gel exclusion on Sephadex G-50 column. It is stable at neutral and alkaline pH and is relatively thermostable. AmI inhibits trypsin and chymotrypsin from the digestive system of insects such as Tribolium castaneum and Locusta migratoria, supporting the hypothesis that inhibitors may have evolved as defense mechanisms of seeds against insects. AmI lost its inhibitory activities when submitted to limited proteolysis with trypsin, while limited proteolysis with chymotrypsin had almost no effect. The partial amino acid sequence of 45 amino acids from the amino terminus of AmI differs significantly from the known sequences of legume-seed and cereal-grain protease inhibitor families. Differences in the chemistry at the inhibitory site(s) and in the amino acid sequence of AmI in comparison to that of other cereal and legume inhibitors suggest that AmI is a member of a new family of serine protease inhibitors. AmI was found to inhibit the anchorage-independent growth of MCF-7 breast cancer cells, suggesting that AmI may have anticarcinogenic activity.

Uptake of human immunodeficiency virus envelope protein gp120 by human trophoblast in culture.

OBJECTIVE: Our purpose was to determine whether human trophoblast has a cell surface CD4 antigen that will bind to gp120, the envelope protein of human immunodeficiency virus. STUDY DESIGN: Uptake of iodine 125-labeled gp120 by trophoblast in culture was measured. Particular attention was paid to technical details that may have caused the contradictory results reported by previous investigators: the source of the recombinant gp120, the method of radioiodination, and the isolation procedure of trophoblast to ensure elimination of contaminating cells, particularly macrophages. RESULTS: Uptake of transferrin-free iodine 125-labeled gp120 to trophoblast was unaffected by adding a 200 molar excess of gp120, by preincubating gp120 with soluble CD4 to block the CD4 binding sites on gp120 and by preincubation of trophoblast with a blocking antibody to CD4 (OKT4a). In contrast, uptake of gp120 by CD4-positive H9 human lymphocytes was reduced 79% by a 200 molar excess of gp120 and > 50% by a CD4-blocking antibody. CONCLUSIONS: Uptake of gp120 to trophoblast is by a high capacity, CD4-independent mechanism that is probably nonspecific and may be related to the mechanism for binding other circulating glycoproteins in maternal blood.

Molecular cloning and identification of a rat serum carboxylesterase expressed in the liver.

We have cloned and sequenced a carboxylesterase from rat liver and purified the corresponding protein from rat blood. The cDNA encodes the entire mature serum esterase protein. It is apparently identical to cDNAs cloned from rat liver by several groups (Long, R. M., Satoh, H., Martin, B. M., Kimura, S., Gonzales, F. J., and Pohl, L. R. (1988) Biochem. Biophys. Res. Commun. 156, 866-873; Takagi, Y., Morohashi, K.-i., Kawabata, S.-i., Go, M., and Omura, T. (1988) J. Biochem. (Tokyo) 104, 801-806; and Robbi, M., and Beaufay, H. (1992) Biochem. Biophys. Res. Commun. 183, 836-841). However, the identification of the protein encoded by this cDNA has not been previously reported. The COOH-terminal -TEHT sequence found in the rat serum carboxylesterase does not possess retention properties and is therefore responsible for its secretion and presence in the circulation. The rat serum carboxylesterase was purified to apparent homogeneity by affinity chromatography on immobilized antibody to rat liver microsomal acyl-CoA thioesterase followed by ion exchange chromatography. The purified protein, with a M(r) of approximately 70,000, was cleaved in situ in a polyacrylamide gel with trypsin, and two peptides were isolated and sequenced. Sequence analysis showed that both peptides were identical only to the corresponding deduced amino acid sequence of the cloned cDNA. Antibodies raised to the COOH-terminal amino acid sequence deduced from the cDNA cross-reacted with the purified rat serum carboxylesterase. Changes in serum esterase activity levels followed changes in protein mass in rat serum and changes in liver mRNA levels in response to various nutritional conditions while total liver esterase activity was essentially unchanged. The above experiments confirm the identity of the protein isolated from rat sera with the cDNA cloned from rat liver and suggest a function for the serum esterase in lipid metabolism.

Synthesis of alpha 1-antichymotrypsin and alpha 1-antitrypsin by human trophoblast.

alpha 1-Antichymotrypsin (alpha 1-ACHY) and alpha 1-antitrypsin (alpha 1-AT) are closely related glycoprotein protease inhibitors, present in plasma and other extracellular fluids, that neutralize proteases released by leukocytes in response to trauma and inflammatory stimuli. Both inhibitors are synthesized primarily by hepatocytes, although lower levels of synthesis by monocytes and breast and intestinal epithelial cells have been demonstrated. Recently, the immunohistochemical localization of alpha 1-AT and alpha 1-ACHY in intrauterine and extrauterine human trophoblastic tissue has been reported. In the present study, we have sought to determine whether human trophoblast is also able to synthesize alpha 1-AT and alpha 1-ACHY. Messenger RNA for both inhibitors was found by Northern blotting in chorionic villi obtained from first trimester and term placenta. Substantial differences in messenger levels for both inhibitors among individual placentas were noted. alpha 1-ACHY and alpha 1-AT messenger was also present in trophoblast cells in primary culture. Synthesis of alpha 1-AT and alpha 1-ACHY protein was demonstrated by SDS-PAGE after immunoprecipitation of [35S]-labeled alpha 1-AT and alpha 1-ACHY from conditioned media of trophoblast cells in culture metabolically labeled with [35S]-methionine. It is of some interest that the M(r) of the alpha 1-AT and alpha 1-ACHY secreted by trophoblast were 50,000 and 49,000, respectively, compared with 54,000 and 68,000 for these proteins in plasma (or secreted by HepG2 human hepatoma and MCF-7 human breast cancer cells).(ABSTRACT TRUNCATED AT 250 WORDS)

alpha 1-Antitrypsin- and anchorage-independent growth of MCF-7 breast cancer cells.

alpha 1-Antitrypsin (alpha 1-AT) and alpha 1-antichymotrypsin (alpha 1-ACHY) are closely related protease inhibitors, synthesized primarily by the liver, which play major roles in modulation of the inflammatory response. Previously, we had shown that MCF-7 human breast cancer cells were able to synthesize active alpha 1-AT and alpha 1-ACHY and that the synthesis of both inhibitors varied among different MCF-7 sublines. We now show that when MCF-7(ML) cells (a subline synthesizing low levels of alpha 1-AT) are grown in soft agar in medium depleted of its trypsin inhibitory capacity (i.e. alpha 1-AT-free), addition of alpha 1-AT (50 micrograms/ml) significantly reduces colony formation in both the presence and absence of estradiol (34% and 44%, respectively). Under these conditions, incubation with 10(-7) M estradiol alone increased colony formation 2- to 3-fold. Colony formation was also significantly reduced by serum leukocyte protease inhibitor, which, like alpha 1-AT, is a potent inhibitor of elastase-like enzymes. We also found that a variety of inflammatory mediators, cytokines, and steroid hormones are able to stimulate synthesis of alpha 1-AT and alpha 1-ACHY by MCF-7 cells. Stimulation by interleukin-6 (IL-6; 200 U/ml), epidermal growth factor (4 nM), and estradiol (10(-7) M) was 2- to 3-fold, whereas stimulations by tetradecanoylphorbol-13-acetate (TPA; 80 nM) and IL-1 (10 U/ml) were 2- to 5-fold and 5- to 10-fold, respectively. In each instance, protein synthesis, monitored by immunoprecipitation of 35S-labeled proteins followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and steady state mRNA levels, monitored by Northern blot analysis with specific cDNA probes, increased to the same extent. Consistent with their ability to stimulate alpha 1-AT synthesis, TPA and IL-1 reduced colony formation in the absence of estradiol by 65% and 63%, respectively. In addition, the effects of both TPA and IL-1 could be reversed by antibody to alpha 1-AT. These results suggest that local synthesis of alpha 1-AT and possibly other protease inhibitors may be important in regulating the tumorigenic potential of MCF-7 breast cancer cells.

Esterase activity in human breast cyst fluid: associations with steroid sulfates and cations.

Human breast cyst fluid (BCF) contains an esterase that on the basis of electrophoretic mobility and response to inhibitors differs from those found in the plasma. From a total of 384 BCF samples analyzed for esterase using p-nitrophenyl hexanoate as substrate, 149 (39%) showed significant activity. The samples had been analyzed for the concentrations of the sulfates of estrone, estriol, dehydroepiandrosterone, as well as the potassium and sodium cations (K+/Na+). The data were submitted to statistical analysis using the Spearman rank order test. The esterase-positive samples exhibited a significant positive association with each of the steroid sulfates and the K+/Na+ ratios. Except for protein concentration, there was no significant correlation between the esterase-positive and esterase-negative cysts. These observations may have physiological significance in that high K+/Na+ ratio cysts have been related to the histological status of the cyst.

Esterase-1: developmental expression in the mouse and distribution of related proteins in other species.

Esterase 1 (Es-1) is a sexually dimorphic 65-kDa glycoprotein present in plasma and other murine tissues able to hydrolyze a variety of esters including fatty acid esters of estradiol. Like most other carboxylesterases, its function is unknown. To gain insight into the function of Es-1 and by analogy other carboxylesterases, we have examined the developmental regulation of Es-1 in the mouse and have looked for the presence of related proteins in the plasma of other species. Northern blot analysis of total RNA from the livers of mice of various ages using a 32P-labeled 470-bp Es-1 cDNA probe showed clear postpartum induction with no detectable Es-1 mRNA in fetal liver. Similarly, immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an affinity-purified rabbit antibody to Es-1 showed no cross-reacting proteins in the plasma until after birth. Northern blot analysis of total RNA from a variety of adult mouse tissues showed the presence of substantial levels of Es-1 mRNA only in liver with lower levels in kidney, testes, and ovaries. Liver mRNA and plasma protein levels rose in parallel attaining full adult levels between 15 and 20 days of age. When plasma proteins were electrophoresed on 7% polyacrylamide gels under nondenaturing conditions, the antibody to Es-1 recognized a low mobility protein in mouse, rat, human, baboon, guinea pig, bovine, horse, and canine but not in chicken plasma. Consistent with the immunoblotting results, the Es-1 cDNA probe hybridized to restriction fragments from human, monkey, rat, and rabbit as well as mouse genomic DNA but not from chicken DNA indicating conservation of the esterase (or esterase-like) gene in mammalian species. The low mobility antigens in mouse and human plasma appeared also to cross-react with antibodies to human thyroglobulin, although antibodies to human thyroglobulin did not appear to recognize Es-1 under these conditions.

Purification and properties of an esterase from human breast cyst fluid.

Levels of estradiol 17 beta-ester hydrolytic activity in the breast cyst fluid (BCF) from 25 different women with fibrocystic disease of the breast were found to vary over a wide range (0-2.4 nmol/min/mg protein for estradiol acetate). On the basis of electrophoretic mobility on agarose gels, the activity from different individuals appeared to be identical. The esterase activity from a single BCF sample was purified to near homogeneity by differential ammonium sulfate precipitation, ion-exchange, and hydrophobic interaction chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, after the final purification step, showed two bands with molecular weights of approximately 22,000 and 23,000, neither of which was immunoreactive with a rabbit antibody raised to a crude esterase-free BCF preparation. Esterase activity could be demonstrated after extraction and renaturation of the protein eluted from the Mr 22,000 band. Resolution of the gel, however, was not good enough to rule out the presence of esterase activity in the Mr 23,000 protein. High performance liquid chromatography gel exclusion chromatography indicated a molecular weight of 90,000-95,000 for the esterase activity in crude BCF and approximately 225,000 for the purified activity, suggesting the native protein to be a tetramer which aggregated during purification. Although the natural substrate of the BCF esterase is unknown, the enzyme is able to cleave a variety of esters including acetate, valerate, and stearate esters of estradiol and p-nitrophenyl hexanoate. It is completely inhibited by diisopropylflurophosphate and diethylnitrophenyl phosphate and partially inhibited by NaF and ebelactone. The substrate and inhibitor profile of the enzyme indicates that it is a "B"-type carboxylesterase and not a protease. A comparison of the properties of the BCF esterase with those of esterases from the formed elements of the blood or from plasma suggests that the BCF esterase is not of blood origin and is probably derived from the cyst itself. Physiologically inactive lipoidal estrogens have been shown to be present in many human body fluids and tissues and it is possible that these esters serve as storage forms of the active hormone in hormonally sensitive tissues where the free steroid could be regenerated by hydrolysis.

Estradiol esters can replace 17 beta-estradiol in the stimulation of DNA and esterase synthesis by MCF-7 cells: a possible role for the estrogen-sensitive MCF-7 cell esterase.

In this communication we extend our earlier observations on estrogen-sensitive carboxyl esterases in MCF-7 human breast cancer cells able to hydrolyze esters of estradiol. Using either estradiol acetate or p-nitrophenyl hexanoate as substrates, esterase activity was found to increase 2-3-fold in MCF-7 cells maintained in the presence of 10(-8) M estradiol. Following sucrose density centrifugation, over 85% of total esterase activity was found in the cytoplasmic fraction. No esterase activity was found in spent media from growing cells. By size exclusion chromatography, estradiol acetate esterase activity exhibited a mol. wt of 45-50 kDa. Attempts to demonstrate incorporation of [3H]estradiol into estradiol fatty acid esters by the above MCF-7 cell line (203P) were unsuccessful, although, such incorporation could be demonstrated in two other MCF-7 cell sublines. Incubation of the 203P cells with 10 nM [3H]estradiol in the presence of 0.5 mM radioinert estradiol acetate resulted in the incorporation of 35 +/- 12% of the label into the estradiol acetate in 10 min. In the absence of radioinert estradiol acetate, no incorporation was observed. When MCF-7 cells were incubated with [3H]estradiol in the presence of a large excess of radioinert estradiol valerate, label was found only in estradiol valerate. Similarly, when the incubation was carried out in the presence of a mixture of radioinert estradiol acetate and valerate, label was incorporated into both esters. We conclude that the apparent formation of radiolabeled estradiol esters by MCF-7 cells incubated under the above conditions, results at least in part, from an esterase-catalyzed exchange reaction. Under conditions where no ester hydrolysis could be detected in the absence of cells, valerate and stearate esters of estradiol were found to be as effective as unesterified estradiol in stimulating esterase synthesis and the incorporation of [3H]thymidine into DNA. These results are consistent with a model in which an intracellular esterase in MCF-7 cells can generate estradiol from an exogenous lipoidal steroid and elicit an estrogen response.

Regulation of antitrypsin and antichymotrypsin synthesis by MCF-7 breast cancer cell sublines.

We have examined the synthesis of the protease inhibitors alpha 1-antitrypsin (alpha 1-AT) and alpha 1-antichymotrypsin (alpha 1-ACHY) by variants of the MCF-7 human breast cancer cell line. Spent medium from MCF-7 203P cells, grown in the absence of serum, was found to contain immunoreactive alpha 1-AT and alpha 1-ACHY by Western blotting. In the presence of 10(-8) M estradiol, levels of both inhibitors were increased 3- to 6-fold. Incubation of spent medium with [125I]trypsin or [125I]chymotrypsin resulted in the formation of stable 75- and 90-kDa complexes identical to the complexes formed between these proteases and the protease inhibitors in plasma, showing the release of active protease inhibitors by MCF-7 cells in culture. Immunoprecipitation of 35S-labeled proteins from the medium of cells grown in the presence of [35S]methionine yielded comparable results, confirming hormonally sensitive synthesis of both protease inhibitors. Northern blot analysis suggests that stimulation of estradiol occurs at the level of transcription. Tetradecanoyl phorbol acetate (50 ng/ml) also stimulated alpha 1-AT and alpha 1-ACHY synthesis 2- to 4-fold, suggesting the involvement of protein kinase-C. Comparison studies with MCF-7 cell sublines ML, BK, 203P, and 300P (a variant spontaneously appearing after 100 passages of 203P) show a wide variation in synthesis of alpha 1-AT and alpha 1-ACHY proteins; sublines 203P and 300P synthesize both inhibitors, the ML subline synthesizes detectable amounts only of alpha 1-ACHY, while no detectable synthesis of either inhibitor was seen in the BK subline. Similar results were obtained for protease inhibitor mRNA transcription by Northern blotting, although low levels of alpha 1-AT mRNA transcription by the ML subline and of alpha 1-AT and alpha 1-ACHY mRNA transcription by the BK subline could be detected.

cDNA cloning of esterase 1, the major esterase activity in mouse plasma.

We report here the cloning of a partial cDNA for Esterase 1, the major esterase activity in mouse plasma. A 470 base pair insert was isolated from a lambda gt11 cDNA library constructed from mouse liver poly A+ RNA, and identified by hybrid selected translation. We show that the sexual dimorphism displayed in the plasma levels of this protein is caused by a difference at the level of transcription. In addition, RFLP data using mouse recombinant inbred strains mapped this clone at the Es-1 locus on mouse chromosome 8.

Effect of warfarin on prothrombin synthesis and secretion in human Hep G2 cells.

Prothrombin synthesis and secretion were studied in a human hepatoma cell line (Hep G2) incubated with 35S-methionine for 2 to 24 hours at 37 degrees C. Extracellular and intracellular prothrombin were detected by immunoprecipitation with affinity-purified antiprothrombin antibody. Incorporation of 35S-methionine into prothrombin was monitored by counting specific bands excised from 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Prothrombin represented 0.3% to 0.7% of total newly synthesized protein secreted into the media. Warfarin had no effect on total prothrombin synthesis (extracellular plus intracellular). However, warfarin inhibited secretion of newly synthesized prothrombin by 58% to 73% over a 2 to 4 hour period. This was accompanied by the intracellular accumulation of an immunoprecipitable species of prothrombin of 78 kd, 6 kd less than extracellular prothrombin. At the end of the 4-hour incubation with warfarin, intracellular prothrombin increased from 44% to 82% (twofold) of total prothrombin, whereas extracellular prothrombin decreased from 56% to 19% (threefold) of total prothrombin. After 24-hour incubation with warfarin, intracellular and extracellular immunoprecipitable prothrombin approached control values. Deglycosylation of extracellular and intracellular prothrombin with hydrofluoric acid (HF) resulted in a decrease in mol wt for both species to 66 kd. Endoglycosidase-H treatment, which digests "early mannosyl" residues, resulted in a decrease in the mol wt of the intracellular species of 8 kd with no effect on the extracellular species. Thus, the lower mol wt intracellular species that accumulates following early warfarin treatment is due to the presence of incompletely processed carbohydrate chain. The data are compatible with the hypothesis that optimum glycosylation and secretion require Vitamin K-dependent carboxylation.

Fetal-specific forms of alpha 1-protease inhibitors in mouse plasma.

Adult mouse plasma contains two distinct alpha 1-protease inhibitors (alpha 1-PIs), which we have called alpha 1-PI(E) and alpha 1-PI(T) because of their differing specificities for elastase and trypsin. Plasma levels of the two mouse inhibitors, determined either functionally or immunologically, are lower in fetal animals than in adults. Western blotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using specific, polyclonal antibodies raised to the inhibitors in adult plasma shows new immunoreactive species of both alpha 1-PI(T) and alpha 1-PI(E) in fetal mouse plasma with apparent molecular weights approximately 1 kDa less than the corresponding proteins in adult plasma. In both cases the fetal alpha 1-PI is replaced by the adult protein between 2 and 6 days after birth. After chemical deglycosylation, the molecular weight difference between the adult and fetal forms of alpha 1-PI(E) and alpha 1-PI(T) is retained, suggesting that the adult and fetal mouse alpha 1-PIs have different amino acid sequences. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protease inhibitors, cleaved specifically at the protease binding site, indicated that the difference in sequence between the adult and fetal proteins resided in the 50-60 kDa N-terminal fragment.