The effect of angiostatic steroids and beta-cyclodextrin tetradecasulfate on corneal neovascularization in the rat.

Folkman and coworkers have described angiostatic steroids that markedly inhibit neovascularization of the rabbit cornea when given topically with beta-cyclodextrin tetradecasulfate (beta-CD), yet have minimal or no glucocorticoid or mineralocorticoid activity. Our objective was to extend these observations to another species, the rat. We induced neovascularization by cauterizing rat corneas with silver nitrate/potassium nitrate; drugs were applied topically four times per day for 4 days in most experiments. Submicron sized emulsions of lipid-soluble dexamethasone and the angiostatic steroids 17 alpha-hydroxyprogesterone (1 or 10 mg ml-1) and cortexolone (1 or 10 mg ml-1) were prepared by lecithin encapsulation of drug microcrystals. The vehicle for water-soluble hydrocortisone 21-phosphate (HCP) +/- beta-CD (Molecusol; Pharmatec, Inc) was 10% Tween 20 in Tris-buffered 0.9% saline. Angiogenesis was significantly inhibited only by 1 mg ml-1 dexamethasone (-63.2% when compared with controls), 0.5 mg ml-1 HCP + 1 mg ml-1 beta-CD (-33.4%), and 1 mg ml-1 HCP (-40.2%). HCP (0.5 mg ml-1) or beta-CD (1 or 2 mg ml-1) alone had no significant effect on neovascularization; the inhibition by 1.0 mg ml-1 HCP was not potentiated by 2 mg ml-1 beta-CD. We also tested HCP and tetrahydro-S (TH-S) using 1.5% hydroxypropyl methylcellulose vehicle and beta-CD from Takeda Chemical Industries, Ltd., to simulate the procedure of Folkman and coworkers.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid sequence determination of ancrod, the thrombin-like alpha-fibrinogenase from the venom of Akistrodon rhodostoma.

The thrombin-like serine protease and antithrombotic agent, Ancrod, was rapidly purified from the crude venom of Akistrodon rhodostoma by agmatine-Sepharose affinity chromatography followed by MonoQ anion exchange chromatography. N-Terminal sequencing and analysis of overlapping proteolytic fragments of purified Ancrod by automated Edman degradation in combination with tandem mass spectroscopy allowed the determination of the 234 amino acid sequence of the protease. Glycosylation sites at all five canonical N-linked glycosylation sites were inferred from the appearance of blank sequencer cycles in the amino acid sequence and were confirmed by mass spectroscopic analysis of the N-glycanase-treated peptides. Monoclonal antibodies raised against the denatured protein and HF-deglycosylated protein recognized Ancrod on Western blots. Sequence comparison to other thrombin-like serine proteases and reptilian fibrinogenases revealed a number of similarities, most notably the catalytic triad and many conserved cysteine positions.

Estrogens reduce bone loss in the ovariectomized, lactating rat model.

Estrogen treatment of ovariectomized, lactating rats improved retention of bone mineral mass by 15-25% compared to ovariectomized, lactating rats receiving vehicle only. On the second day postpartum all lactating rats were ovariectomized and were placed along with age-matched non-mated controls on a whole-wheat flour-based diet with 0.1% calcium and 0.4% phosphorus. On day 6 postpartum estrogen treatment was begun with either implantation of a slow-release 17 beta-estradiol pellet or with the first of daily subcutaneous injections in sesame oil (vehicle). Increasing doses of estrogen resulted in decreased food consumption and decreased litter weight gain, both well-known effects of estrogens. Ovariectomized, lactating rats implanted with a slow-release pellet containing 0.35 mg 17 beta-estradiol had mean serum estradiol levels of 113.5 pg/ml. At the end of 21 days of lactation, femurs of dams with placebo pellets showed loss of 54% of bone ash weight compared with the non-mated controls versus only 42% loss by rats receiving estradiol treatment. Rats were also injected with estradiol benzoate in a sesame oil vehicle at 3 dose levels of 1.6, 5, or 16 micrograms/day. Only the 5 and 16 micrograms/day doses significantly improved retention of bone mineral mass during lactation (+17% and +18%, respectively, vs vehicle-injected, lactating rats). Estrone administered by subcutaneous injection also improved retention of bone during lactation; however, injection of 50 micrograms/day of estrone was required to produce an equivalent bone retention compared to 5 micrograms/day of estradiol. Thus, treatment of ovariectomized, lactating rats with estrogens results in a significant reduction of the loss of bone mineral mass associated with lactation.

Isolation and complete amino acid sequence of two fibrinolytic proteinases from the toxic Saturnid caterpillar Lonomia achelous.

The major toxic and fibrinolytic activity of the saliva and hemolymph of the larval form of Lonomia achelous was purified to homogeneity by a combination of metal chelate and affinity chromatography. Two apparent isozymes, Achelase I (213 amino acids, pIcalc = 10.55) and Achelase II (214 amino acids, pIcalc = 8.51), were sequenced by automated Edman degradation, and their C-termini confirmed by Fourier-transform mass spectrometry. The calculated molecular weights (22,473 and 22,727) correspond well to Mr estimates of 24,000 by SDS-PAGE. No carbohydrate was detected during sequencing. The enzymes degraded all three chains of fibrin, alpha greater than beta much greater than gamma, yielding a fragmentation pattern indistinguishable from that produced by trypsin. Chromogenic peptides S-2222 (Factor Xa and trypsin), S-2251 (plasmin), S-2302 (kallikrein) and S-2444 (urokinase) were substrates while S-2288 (broad range of serine proteinases including thrombin) was not hydrolyzed. Among a range of inhibitors Hg+2, aminophenylmercuriacetate, leupeptin, antipain and E-64 but not N-ethylmaleimide or iodoacetate abolished the activity of the purified isozymes against S-2444. Phenylmethylsulfonyl fluoride, soybean trypsin inhibitor and aprotinin were less effective. The presence of the classic catalytic triad (histidine-41, aspartate-86 and serine-189) suggests that Achelases I and II may be serine proteinases, but with a potentially free cysteine-185 which could react with thiol proteinase-directed reagents.

The ovariectomized, lactating rat as an experimental model for osteopenia: calcium metabolism and bone changes.

The ovariectomized, lactating rat (Sprague-Dawley) is proposed as an experimental model for the rapid development of osteopenia which may be used to test the effectiveness of bone-retentive drugs potentially useful in treating osteoporotic women. Rats were ovariectomized (OVX) on day 2 postpartum and were kept on a low-calcium diet (0.1%). Measurements of serum total calcium, ionic calcium, albumin and parathyroid hormone were conducted between days 4 and 21 of lactation. Serum total and ionic calcium and albumin were significantly lower and serum parathyroid hormone was significantly higher in all lactating rats at 16 days postpartum compared to nonlactating controls. Mean bone mass of the femurs of OVX lactating rats measured at day 21 was approximately 50% of that of non-lactating intact controls. The enhanced duodenal calcium absorption (in everted gut sacs) associated with lactation was not affected by OVX and neither was the average litter weight gain between 2 and 14 days of lactation. In conclusion, lactation coupled with a low-calcium diet resulted in marked osteopenia, depressed serum calcium (both total and ionic) and significantly elevated serum parathyroid hormone concentration. The rapid and extensive bone loss of this model makes it appropriate for the study of therapeutic agents designed to retain bone mass.

Insulinlike growth factor I receptors in rabbit gastrointestinal tract. Characterization and autoradiographic localization.

Insulinlike growth factor I is a potent mitogen with insulinlike metabolic effects. Insulinlike growth factor I is synthesized in the liver, intestine, and other organs. Insulinlike growth factor I receptors are widely distributed and structurally similar to insulin receptors. Frozen sections of rabbit gastrointestinal tract were incubated in buffer containing 40 pmol/L [125I]insulinlike growth factor I. Binding was saturable, temperature- and time-dependent, and reversible. Saturation binding experiments showed a single class of high-affinity receptors (Kd = 0.9 nmol/L, Bmax = 0.36 pmol/mg protein). The IC50s for insulinlike growth factor I and insulinlike growth factor II were 3 nmol/L and 90 nmol/L, respectively; whereas insulin at 1-3 mumol/L displaced 50% of specific binding. Autoradiography of insulinlike growth factor I binding demonstrated significant differences in receptor density in gastrointestinal smooth muscle, epithelium of the esophagus, stomach, small intestine, and colon. These results indicate that a single class of specific, high-affinity insulinlike growth factor I receptors were distributed in muscular and mucosal layers of the entire rabbit gastrointestinal tract. Insulinlike growth factor I is likely to be an important local mediator of intestinal growth and metabolism.

Peroxisome proliferator-binding protein: identification and partial characterization of nafenopin-, clofibric acid-, and ciprofibrate-binding proteins from rat liver.

Peroxisome proliferators (PP) induce a highly predictable pleiotropic response in rat and mouse liver that is characterized by hepatomegaly, increase in peroxisome number in hepatocytes, and induction of certain peroxisomal enzymes. The PP-binding protein (PPbP) was purified from rat liver cytosol by a two-step procedure involving affinity chromatography and ion-exchange chromatography. Three PP, nafenopin and its structural analogs clofibric acid and ciprofibrate, were used as affinity ligands and eluting agents. This procedure yields a major protein with an apparent Mr of 70,000 on NaDodSO4/PAGE in the presence of reducing agent and Mr 140,000 (Mr 140,000-160,000) on gel filtration and polyacrylamide gradient gel electrophoresis under nondenaturing conditions, indicating that the active protein is a dimer. This protein has an acidic pI of 4.2 under nondenaturing conditions, which rises to 5.6 under denaturing conditions. The isolation of the same Mr 70,000 protein with three different, but structurally related, agents as affinity ligands and the immunological identity of the isolated proteins constitute strong evidence that this protein is the PPbP capable of recognizing PP that are structurally related to clofibrate. The PPbP probably plays an important role in the regulation of PP-induced pleiotropic response.

Immunocytochemical demonstration of estrogen receptors by monoclonal antibodies in human breast cancer: correlation with estrogen receptor assay by dextran-coated charcoal method.

Immunocytochemical demonstration of estrogen receptors in 115 human breast cancer specimens was performed using mouse monoclonal antibodies against estrogen receptor and avidin-biotin as the displaying system. The antibody indicated a highly heterogeneous endowment of neoplastic cells with estrogen receptor at both nuclear and cytoplasmic levels. The percentage of labeled cells within each tumor specimen was recorded to compare this immunocytochemical assay with the biochemical assay of estrogen receptors by the dextran-coated charcoal method. A significant correlation was observed between these two assays. The present results show that estrogen receptors can be confidently demonstrated at the single cell level, thus providing additional information to quantitative biochemical assays. Their prognostic and therapeutic predictive powers may be usefully integrated, particularly in view of the heterogeneous distribution of receptors among cancer cells.

Chemical identification of a tumor-derived angiogenic factor.

Neoplasms produce substances that induce blood vessel formation (angiogenesis). Fractions from ethanol extracts of the Walker 256 carcinoma were isolated by silica column chromatography and C18 reversed-phase high-performance liquid chromatography. Two of the isolated fractions induced neovascularization when tested in the rabbit corneal micropocket assay. One of the fractions was identified as nicotinamide by desorption-electron impact mass spectrometry, nuclear magnetic resonance spectroscopy, and gas chromatography-mass spectrometry. The second active fraction contained nicotinamide as part of a more complex, as yet unidentified, molecular arrangement. Microgram quantities of commercial nicotinamide induced neovascularization in the corneal micropocket assay and in the chick chorioallantoic membrane assay.

Estrogenic effect of phenol red in MCF-7 cells is achieved through activation of estrogen receptor by interacting with a site distinct from the steroid binding site.

MCF-7 cells serially subcultured in media containing phenol red show poor stimulation of progesterone receptor (PR) synthesis in response to estradiol compared to cells grown in phenol red-free media. Phenol red, when added to cytosol, did not compete with [3H]estradiol for estrogen binding sites in concentrations ranging from 2 microM-1 mM. However 25 microM of the dye was sufficient to increase nuclear translocation of estrogen receptor (ER) in the intact cell. Phenol red activates cytoplasmic ER as indicated by DNA-cellulose binding studies. When cells grown in phenol red-free medium were exposed to phenol red for 48 h, PR levels increased in a dose dependent manner. From these data, it may be concluded that phenol red causes estrogenic effect in MCF-7 cells through activation of cytoplasmic receptor by interacting at a site distinct from the steroid binding site.

Are estrogen receptors cytoplasmic or nuclear? Some immunocytochemical and biochemical studies.

The subcellular localization of estradiol receptor (ER) has been examined using various experimental approaches. Immunocytochemical studies using the monoclonal antibody JS 34/32, raised against calf uterine cytosolic ER, yielded only equivocal results. In general, cells and tissues pretreated with estradiol showed positive immunostaining in the nuclei whereas those not exposed to the steroid did not show any staining. Nuclear translocation of ER was examined in intact MCF-7 cells using compounds which are known to influence receptor activation. When MCF-7 cells were exposed to molybdate (20 mM), nuclear translocation was completely inhibited while dithiothreitol (20 mM), dibutyryl cAMP (1 microM) and dibutyryl cGMP (1 microM) increased the translocation 2-3-fold. Phenol red, at the range of concentrations generally used in tissue culture media, also increased translocation. The physiological validity of such translocation was examined using cellular progesterone receptor (PR) synthesis as a specific parameter. When MCF-7 cells were grown in media containing phenol red for 48 h, the PR synthesis increased significantly. We further examined whether cytoskeletal proteins are involved in the translocation of ER. Colchicine, an inhibitor of microtubule assembly, inhibited translocation of ER in MCF-7 cells at 1-10 microM. PR synthesis was also inhibited by colchicine in a dose-dependent manner. It may be concluded from these and other published data that ER may not be located at all times in a single subcellular compartment but may rather exist in a dynamic equilibrium between the plasma membrane, cytoplasm and nucleus.

Immunohistochemical localization of estrogen receptor in rat brain, pituitary and uterus with monoclonal antibodies.

Localization of estrogen receptor (ER) in rat brain, pituitary and uterus is shown by the avidin-biotin complex technique using a monoclonal antibody, JS34/32. Immunostaining is observed in nuclei of certain neurons in the preoptic-septal region, hypothalamus and amygdala, and in cells of the anterior pituitary and uterus after estradiol stimulation. Staining is specific since preadsorbed JS34/32 antibody with purified cytoplasmic ER as well as a control monoclonal antibody do not show positive immunoreaction. In the brain, neither cytoplasmic nor nuclear staining is seen in the absence of estradiol stimulation, nor with the progesterone and dihydrotestosterone treatments. The distribution of ER-containing neurons in specific areas of the brain overlaps with the distribution of estrogen target neurons demonstrated by autoradiography. The results demonstrate the usefulness of the monoclonal antibody for the detection of ER in target tissues.

Interaction of estrogen receptor of calf uterus with a monoclonal antibody: probing of various molecular forms.

A monoclonal antibody to estrogen receptor (JS34/32) is able to recognize, in the calf uterine cytosol, a protein (approximately 65 000 daltons) giving a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two molecules of this antibody are able to simultaneously interact with the native 8S form of the receptor present in the calf uterine cytosol ("twin antibody" assay). This indicates the presence of two antigenic determinants on the "low-salt" 8S form of the receptor. This form of the receptor shows an increase in Mr from 345 000 to 665 000 after interaction with the soluble antibody. Dissociating agents that induce the dissociation of the 8S form to smaller forms also induce the dissociation of the two antigenic determinants. The 4S "high-salt" form of the estrogen receptor has one determinant per molecule, appearing to be the smallest form of the receptor not containing repetitive structures associated with the steroid binding site. The nuclear receptor also shows the presence of more than one antigenic determinant on its molecule.

Binding of monoclonal antibodies to the nuclear estrogen receptor in intact nuclei.

A monoclonal antibody to calf uterus cytoplasmic estrogen receptor shows a specifically displaceable and saturable binding to intact nuclei of mouse uterus after estradiol stimulation. The binding is complete after 3 hr at 0 degree C. The binding of the antibody correlates with the exchangeable estradiol binding activity of the nuclei over a 4-hr time course following in vivo injection of 17 beta-estradiol.

Monoclonal antibodies against estrogen receptor: interaction with different molecular forms and functions of the receptor.

Hybridoma cells have been produced by fusing SP2/O-Ag14 mouse myeloma cells with spleen cells from a mouse immunized with a purified preparation of estrogen receptor from calf uterus. The antibodies, all of the immunoglobulin G (IgG) class, interact with different forms of calf receptor as well as with rat and human receptors. The equilibrium dissociation constant of the antibody-receptor complex was measured in solid phase and in solution. With immobilized antibodies the Kd is 0.06 nM whereas in solution it is 0.5 nM. Only one antigenic determinant is present per molecule of receptor with the antibodies tested. The antibodies JS34/32 are able to form only a 1:1 complex with the 8S form of the receptor, whereas a 2:1 receptor-IgG complex is formed at low antibody concentration with the high-salt or nuclear form of receptor. The antibodies JS34/32 and JS28/32 prevent neither the nuclear uptake of the receptor nor the extraction of the translocated receptor from the nuclei.

Effect of sodium molybdate on cytosolic estrogen receptor.

Estrogen binding activity of crude calf uterus cytosol is rapidly destroyed in heating. The time course of inactivation at 37 degrees C shows a biphasic pattern; sodium molybdate (5-10 mM) completely blocks one of the components in the estradiol-free cytosol, while it has little effect on cytosolic receptor complexed with estradiol. Partially purified native 8S receptor loses its heat sensitivity, and, as a consequence, the molybdate effect disappears. By sucrose gradient analysis of crude cytosol it is evident that molybdate does not affect the sedimentation properties of the estradiol receptor at low temperature. However, at increasing temperatures, molybdate prevents the disappearance of the receptor peak in the crude cytosol or the formation of large, KCl-resistant, aggregates in the presence of estradiol. The partially purified native 8S receptor does not aggregate on heating; addition to it of receptor-depleted cytosol results in the recovery of heat inactivation and aggregate formation, and this is prevented by molybdate. Molybdate has no protective effect on any other inactivating agent which does not act through aggregation of receptors. A crude cytosolic preparation of the receptor which is unable to form heat-dependent aggregates does not display the fast heat inactivating component.

Sepharose-immobilized triazine dyes as adsorbants for human lymphoblastoid interferon purification.

An extensive selection of immobilized triazine dyes have been examined for their potential as adsorbants for human lymphobalstoid interferon. Procion red HE7B was selected as the most suitable for preparative scale purification. Sepharose-immobilized procion red HE7B is able to bind 10(5) reference units/mL of interferon from cell supernatants and can be eluted with at least 25-fold purification and 90% yield by a KC1 gradient. Further purification was obtained either by reapplying the eluted interferon after dialysis to the dye column or by gel filtration on Ultrogel AcA 34 after lyophilization and dialysis. The latter procedure gave a final activity of about 10(6) U/mg protein and approximately 75% recovery of interferon activity.

Identification of high affinity estrogen binding sites in calf uterine microsomal membranes.

Membrane-associated binding sites with high affinity and specificity for estrogens have been identified in calf uterine microsomes. The binding of 17 beta-[3H]estradiol is specific and saturable at low hormone concentration (2 nM) of high affinity (Kd = 0.5 nM) and sensitive to trypsin and other proteolytic enzymes. Binding of [3H]estradiol to membranes is inhibited by low concentrations of unlabeled 17 beta-estradiol and diethylstilbestrol (50 to 100 pM) while high concentrations of nonestrogenic steroids have little effect. The nondisplaceable binding is low and never exceeds 15% at the half-maximal point of specific binding. The maximum amount of ligand bound per mg of membrane protein is in the range of 0.4 to 1.0 pmol. Specific estradiol binding associated with microsomal fractions varies between 7 to 15% of the total binding sites. Estradiol appears not to be metabolized to any significant extent after binding to uterine membranes. Whereas the affinities of the estrogens tested are similar, the affinity of the antiestrogen, Tamoxifen, for the cytosolic receptor is at least 10 times higher than for the microsomal binding sites. In contrast to rat uterus and ovaries, the microsomal membranes from various nontarget rat tissues do not show any specific binding.

Quantitation of methionyl peptides in nanomole quantities by a fluorometric method.

A quantitative and highly specific method to determine low concentrations of methionyl peptides, which do not contain tryptophan or cysteine residues, has been developed. The method is based on the stoichiometry and selectivity of N-chlorosuccinimide (NCS) towards methionine and N-acetyltryptophan. N-Chlorosuccinimide reacts with N-acetyltryptophan in a 1:1 ratio to produce the N-acetyl-2-oxindolealanine--a derivative essentially devoid of fluorescence. The decrease in fluorescence intensity is approximately linear with respect to the NCS concentration. Preincubation of NCS with methionine or methionyl peptide consumes a stoichiometric amount of the reagent and the unreacted NCS is quantitated by the decrease in fluorescence intensity resulting upon incubation of the mixture with 1 eq of N-acetyltryptophan. Less than 1 nmol of methionyl peptide can be accurately quantitated by this method.