Inhibition of fibrinogen receptor expression and serotonin release by leupeptin and antipain

Human platelet agonists such as thrombin, ADP, and collagen stimulate the rapid expression of fibrinogen receptors. In other cell types, calcium-activated proteases have been suggested to participate in the mechanism of expression of cell surface receptors (Lynch, G., and Baudry, M. (1984) Science 224, 1057-1063). In platelets the majority of the neutral protease activity is calcium-activated protease. We examined the effects of leupeptin and antipain, two calcium-activated protease inhibitors, on the expression of platelet fibrinogen receptors. These inhibitors abolished thrombin and ADP-induced fibrinogen binding. This inhibition required the addition of leupeptin or antipain prior to the agonist and was not due to displacement of fibrinogen from its receptor or inhibition of agonist binding to platelets. Leupeptin and antipain also inhibited fibrinogen-independent thrombin-stimulated release of serotonin. These results are discussed in relation to the involvement of calcium-activated protease in early events of platelet activation.     

Activation and Inhibition of Cerebral Prolidase

Purified of prolidase from calf brain (acetone and [NH4]2SO4 fractionation) separated this enzyme from proteases, leucine aminopeptidase, master dipeptidase, and Gly-Gly dipeptidase. Prolidase was tested with peptidase and protease inhibitors, used at higher levels (35 times or more) than their ID50 for peptidases and proteases. Bacitracin, leupeptin, chymostatin, and antipain had no effect; pepstatin slightly increased activity, and only bestatin was inhibitory. Antibiotics that affect protein synthesis did not inhibit prolidase. Peptides with proline at the NH2 end activated prolidase, whereas those with proline at the carboxyl end inhibited it. Di, tri, and tetra-Pro peptides increased prolidase activity. Thyrotropin-releasing hormone had no effect on prolidase; its analog Pro-His-Pro-NH2 gave high activation and decreased the Km from 20 mM to 1.54 mM. Pro-peptide inhibitors and activators were not themselves split by prolidase. The results indicate influences of specific peptides, for both inhibition and activation, on prolidase activity.     

Reduction in cell entry of Eimeria tenella (Coccidia) sporozoites by protease inhibitors, and partial characterization of proteolytic activity associated with intact sporozoites and merozoites

The role of proteases in the invasion of host cells by Eimeria tenella (Wisconsin strain) was studied in vitro. Protease inhibitors were used to treat sporozoites before inoculation or were applied to cultured chicken kidney cells before infection. The inhibitors antipain, leupeptin, aprotinin, L-1-tosylamide-2-phenyl-ethyl chloromethyl ketone (TPCK), or N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) reduced parasite invasion to 16-66% of control after treatment of cultured cells or sporozoites with 5- or 50-micrograms/ml concentrations of inhibitors in the culture medium. Phenylmethylsulfonyl fluoride (PMSF) reduced invasion to 32-57.7% at concentrations of 1-4 mM. The optimum pH for hydrolysis of azocasein by intact sporozoites or merozoites was determined over a range of pH 5.0 to pH 9.0. Sporozoites were highly active over a broad range from pH 5.5 to pH 9.0, with an apparent optimum at pH 8.0. Merozoites had a much lower specific activity with pH optima at 7.0 and 8.5. The protease activity of sporozoites or merozoites could be inhibited completely by the addition of 50 micrograms/ml of leupeptin, TPCK, or TLCK or of 4 mM PMSF. Antipain inhibited proteases of sporozoites but not of merozoites. Pepstatin had little effect on either sporozoites or merozoites. The results suggest that parasite proteases of Eimeria may be necessary for invasion of host cells.     

The non-DNA-binding heterooligomeric form of mammalian steroid hormone receptors contains a hsp90-bound 59-kilodalton protein

Untransformed cytosol receptors for progesterone (PR), androgen (AR), estrogen (ER), and glucocorticosteroid (GR) in rabbit tissues contain a 59-kDa protein (p59) (Tai, P.K.K., Maeda, Y., Nakao, K., Wakim, N.G., Duhring, J.L., and Faber, L.E. (1986) Biochemistry 25, 5269-5275) and a 90-kDa heat shock protein (hsp90). In the present study, receptors from calf uterus (PR, AR, ER, and GR) and from human breast cancer MCF7 cells (PR and GR) were also shown to be comprised of hsp90 and p59. These heterooligomer receptor complexes were stabilized both by transition metal oxyanions (molybdate and tungstate) and chemical cross-linking with dimethylpimelimidate. In 0.4 M KCl, tungstate-stabilized (but not molybdate-stabilized) PR, AR, ER, and GR retained hsp90, but lost p59. Dimethylpimelimidate cross-linking prevented p59 dissociation from hsp90-receptor complexes. Stabilization with tungstate and/or cross-linking permitted immunoaffinity purification of untransformed rabbit as well as calf PR and ER on EC1-Affi-Gel 10 column (an anti-p59 immunoadsorbant). Combined immunoaffinity purification and cross-linking experiments indicated that p59 is bound to hsp90 in the cytosol. We propose that in the nontransformed steroid receptor, p59 interacts with hsp90 rather than with the hormone binding subunit.     

Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient

A procedure for rapid, preparative purification of plasmid DNA is described and compared with a conventional equilibrium centrifugation method. A discontinuous, two-step CsCl-ethidium bromide gradient is used, with the starting position of the plasmid-containing extract being at the bottom of the tube. During centrifugation in a fixed angle rotor, covalently closed circular plasmid DNA is separated from contaminating protein, RNA, and chromosomal DNA in 5 hr. Plasmids purified by this method are considerably less contaminated with RNA than when purified by a 48-hr equilibrium run in a homogeneous gradient, as determined by agarose gel electrophoresis and 5'-end-labeling studies. Plasmid DNA purified in two-step gradients can be used directly for restriction endonuclease analysis and DNA sequencing.     

Inactivation of porcine corpus luteum lutropin receptors by a microsomal enzyme: Activation in ageing and regressing corpora lutea

The binding of ¹²⁵I-labelled human choriogonadotropin (hCG) to homogenates of porcine corpora lutea showed a marked departure from ideal behaviour, due to a time- and temperature-dependent inactivation of both free hormone and unoccupied receptors. Occupied receptors were not affected. Lutropin (LH)-receptor-inactivating activity was detected after preincubation of homogenates, particulate fractions and microsomes, but little activity could be demonstrated in cytosol fractions. Inactivation was dependent on the temperature and pH of preincubation, and on tissue concentration: LH-receptor inactivation was first-order with respect to preincubationtime. Lutropin-receptor-inactivating activity was low in early-luteal and mid-luteal phase in pig corpora lutea, but was increased significantly in late-luteal and regressing corpora lutea.     

Inhibition of thrombin-induced platelet activation by leupeptin. Implications for the participation of calpain in the initiation of platelet activation

Inhibitors of calcium-dependent proteases (calpains) such as leupeptin and antipain have been shown to selectively inhibit platelet activation by thrombin. Based upon this observation, it has been proposed that calpains play a role in the initiation of platelet activation. In the present studies, we have examined the effect of leupeptin on the earliest known event in thrombin-induced platelet activation: the interaction between the agonist, its receptors, and the guanine nucleotide-binding proteins which stimulate phospholipase C (Gp) and inhibit adenylyl cyclase (Gi). We found that leupeptin inhibited thrombin's ability to stimulate phosphoinositide hydrolysis, suppress cAMP formation, and dissociate Gp and Gi into subunits. Leupeptin had no effect, however, on the same responses to other agonists or on thrombin binding to platelets. Although these observations might suggest, as others have concluded, that calpain is involved in the initiation of platelet activation by thrombin, we also found that: 1) substituting platelet membranes for intact platelets and decreasing the free Ca2+ concentration below the threshold required for calpain activation did not diminish the effects of leupeptin on phosphoinositide hydrolysis and cAMP formation, 2) washing the platelets after incubation with leupeptin reversed the effects of the inhibitor, 3) permeabilizing the platelets with saponin did not enhance the inhibitory effects of leupeptin, and 4) leupeptin inhibited the proteolysis of fibrinogen and the hydrolysis of S2238 by thrombin. Similar results in these assays were obtained with antipain. Therefore, our observations suggest that the inhibition of platelet activation by leupeptin is due to a direct interaction with thrombin and need not reflect a role for calpain in the initiation of platelet activation.     

CAR and PXR: the xenobiotic-sensing receptors

The xenobiotic receptors CAR and PXR constitute two important members of the NR1I nuclear receptor family. They function as sensors of toxic byproducts derived from endogenous metabolism and of exogenous chemicals, in order to enhance their elimination. This unique function of CAR and PXR sets them apart from the steroid hormone receptors. In contrast, the steroid receptors, exemplified by the estrogen receptor (ER) and glucocorticoid receptor (GR), are the sensors that tightly monitor and respond to changes in circulating steroid hormone levels to maintain body homeostasis. This divergence of the chemical- and steroid-sensing functions has evolved to ensure the fidelity of the steroid hormone endocrine regulation while allowing development of metabolic elimination pathways for xenobiotics. The development of the xenobiotic receptors CAR and PXR also reflect the increasing complexity of metabolism in higher organisms, which necessitate novel mechanisms for handling and eliminating metabolic by-products and foreign compounds from the body. The purpose of this review is to discuss similarities and differences between the xenobiotic receptors CAR and PXR with the prototypical steroid hormone receptors ER and GR. Interesting differences in structure explain in part the divergence in function and activation mechanisms of CAR/PXR from ER/GR. In addition, the physiological roles of CAR and PXR will be reviewed, with discussion of interactions of CAR and PXR with endocrine signaling pathways.     

Hydrolysis of soybean trypsin inhibitor by the gamma subunit of 7SNGF and EGF-BP

Although soybean trypsin inhibitor (STI) does not inhibit the esterase activity of either epidermal growth factor binding protein (EGF BP) or the gamma subunit of 7SNGF, it does behave as a substrate for proteolysis. Cleavage of the active site peptide bond of STI does occur when incubated in the presence of either EGF-BP or the gamma subunit of 7SNGF. The hydrolysis id pH dependent with maximum proteolysis at pH 6.0-7.0. the newly formed C-terminal arginine residue in modified STI can be released by carboxypeptidase B digestion. Both enzymes are inhibited by low concentrations (2-4 microgram/ml) of the microbial protease inhibitors leupeptin and antipain. These inhibitors are specific for trypsin-like proteases. Since both enzymes can be found as part of high molecular weight complexes with growth factors these results confirm the hypothesis that they are involved during a postranslational modification event.     

Inhibition of starfish oocyte maturation by some inhibitors of proteolytic enzymes

Starfish oocyte maturation is triggered by a natural hormone, 1-methyladenine (1-MeAde), produced in the follicle cells, or artificially by dithiothreitol (DTT). These substances act on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), which induces germinal vesicle breakdown (GVBD) and subsequent processes of meiotic maturation. Further, MPF is amplified in immature oocytes that have received the injection of MPF. In this paper the effect of leupeptin and antipain, protease inhibitors of microbial origin, on starfish oocyte maturation was investigated. The protease inhibitors were found to inhibit 1-MeAde-induced maturation when they were applied externally or injected into oocytes. DTT-induced maturation was also inhibited by injection of leupeptin. However, leupeptin did not inhibit the maturation-inducing action of MPF or MPF amplification. These results show that the protease inhibitors suppress the production of MPF by 1-MeAde or DTT, suggesting that some endogenous protease(s) acts in the production of MPF.     

Immunochemistry for oestrogen receptor,progesterone receptor and HER2 on cell blocks in primary breast carcinoma

K. Kumar S, N. Gupta, A. Rajwanshi, K. Joshi and G. Singh Immunochemistry for oestrogen receptor, progesterone receptor and HER2 on cell blocks in primary breast carcinoma Objective: Steroid receptors and human epidermal growth receptor 2 (HER2) have been used for predicting response to treatment in breast cancers. Fine needle aspiration cytology can provide highly cellular material and can be used for such analysis. The present study was undertaken to assess the reliability of oestrogen and progesterone receptor (ER, PR) status and HER2 as demonstrated by immunochemistry (IHC) on cell blocks from breast carcinoma cases, in comparison with histological sections. Methods: IHC for ER, PR and HER2 was performed on cell blocks and their corresponding tissue sections of 50 primary pre‐chemotherapy breast carcinomas. Positivity for ER and PR was scored according to the Allred scoring system. Strong membranous positivity in more than 30% of tumour cells was considered positive for HER2. The tumours were classified as luminal A, luminal B, HER2‐over‐expressing and triple negative on the basis of ER, PR and HER2 status and results on cell blocks compared with histological sections. Results: Correlation between immunostaining on cell blocks and the corresponding tumour tissues revealed a concordance rate for ER, PR and HER2 of 90% [Correlation coefficient (r) = 0.79], 94% (r = 0.86) and 90% (r = 0.76), respectively. Including five cases in which cell blocks were either ER or PR positive, 43/50 cases (86.0%) could be correctly classified on cell block immunostaining alone. The main reasons for seven discordant cases included technical errors (sampling error and staining error) and interpretational error in HER2 evaluation on cell blocks; the core biopsy was inadequate in one, and apparently false negative for HER2 in another. Conclusion: Cell blocks are useful in the assessment of hormone receptor status and HER2 by IHC, especially in cases of locally advanced breast cancer for planning neoadjuvant chemotherapy. It is highly recommended to have good quality cell blocks and quality control of their interpretation.     

Detection and characterization of a selective endopeptidase from Plasmodium berghei by using fluorogenic peptidyl substrates

By using fluorogenic peptidyl-3-amino-9-ethyl-carbazole a highly selective endopeptidase for the Val-Leu-Gly-Arg sequence was demonstrated in endoerythrocytic stages of $\text{Plasmodium berghei}$. Val-Leu-Gly-Arg-endopeptidase showed a maximum activity in pH range 7.0–8.0; it was completely inhibited by 1 mM leupeptin and 1 mM antipain. A complete inhibition was also obtained by 15 mM chloroquine. This trypsin-like activity was negligible in uninfected red blood cells. The high sensitive fluorogenic procedure could be performed on cell fractions, cell lysates as well as supernatants.     

Challenges to defining a role for progesterone in breast cancer

Progesterone is an ovarian steroid hormone that is essential for normal breast development during puberty and in preparation for lactation. The actions of progesterone are primarily mediated by its high affinity receptors, including the classical progesterone receptor (PR) -A and -B isoforms, located in diverse tissues such as the brain where progesterone controls reproductive behavior, and the breast and reproductive organs. Progestins are frequently prescribed as contraceptives or to alleviate menopausal symptoms, wherein progestin is combined with estrogen as a means to block estrogen-induced endometrial growth. Estrogen is undisputed as a potent breast mitogen, and inhibitors of the estrogen receptor (ER) and estrogen producing enzymes (aromatases) are effective first-line cancer therapies. However, PR action in breast cancer remains controversial. Herein, we review existing evidence from in vitro and in vivo models, and discuss the challenges to defining a role for progesterone in breast cancer.     

Expression of oestrogen receptor-beta in invasive breast tumours

Steroid hormone receptors are used routinely to predict endocrine responsiveness in patients with breast cancer. Two oestrogen receptors (ERs): ER alpha and ER beta have been identified. Although ER alpha and ER beta genes share a large degree of homology, it is generally thought that their distribution and function are substantially different in many tissues. Both of them may be expressed in normal and neoplastic tissues of the breast. While much is known about ER alpha, the role of ER beta is still undefined, especially at the protein level. Recent development of reliable antibodies to ER beta has provided opportunity to test immunohistochemical reactions detecting ER beta in archival breast tumours. The aim of our study was to learn more about the cellular mechanisms underlying the relationship of ER beta and progesterone receptor (PR) in breast cancer tissues, discriminating between hormone-dependent and hormone-independent tumours. ER alpha and PR content of tumour tissues of 154 patients with breast cancer were tested by in situ indirect immunohistochemical method parallel with ligand binding biochemical assay. ER beta was detected in 8 ER alpha-/PR+ breast carcinomas by immunohistochemical method too. Steroid hormone receptor content was analysed comparing to the histologic type and grading of the tumours. CONCLUSIONS: A considerable part of breast carcinomas belongs to the ER+/PR+ and ER-/PR- groups. About 1-2% of the tumours is expected to be ER alpha-/ER beta+/PR+ type. In such cases ER alpha negative reaction together with PR positivity can signal the necessity of the immunohistochemical detection of ER beta in routine histopathological practice, presenting the precise steroid hormone receptor status for the most effective endocrine therapy of the patients.     

Degradation of Rat Brain Cholinergic Muscarinic Receptors In Vitro: Enhancement by Agonists and Inhibition by Antagonists

Cholinergic muscarinic receptors undergo proteolytic degradation in vitro under physiological conditions as shown by a loss in [3H]quinuclidinylbenzilate binding activity. The serine protease inhibitor phenylmethylsulfonyl fluoride was very effective in diminishing the receptor loss. Soybean trypsin inhibitor was less effective. Both EDTA and EGTA were also effective in abolishing receptor degradation, suggesting the involvement of metallopeptidases in the process. Calcium-dependent neutral proteases requiring sulfhydryl reducing agents did not seem to be involved in receptor degradation. Dithiothreitol failed to enhance receptor degradation and iodoacetamide, leupeptin, and antipain, inhibitors of this enzyme class, failed to alter receptor loss as measured by radioligand binding. Most of the proteolytic activity occurred in the cytosol and was readily resolved from the receptor in the membrane fraction. We found that [3H]quinuclidinylbenzilate, an antagonist, inhibited the rate of receptor loss. On the other hand, agonists (acetylcholine, methacholine, and muscarine) appeared to enhance the rate of receptor loss. We postulate that these opposite effects are due to differences in receptor conformation in response to ligand binding. Susceptibility to proteolysis may therefore serve as a probe for receptor conformation.