Search

Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.

In order to investigate the relationship between the increment of plasma prolactin (PRL) levels and the change of plasma levels of PRL, DHEA-S, cortisol, aldosterone and 17 alpha OH delta 5-P were quantified by respective RIA in patients treated with TRH parenterally or with sulpiride orally. The results were as follows: During the infusion of TRH, plasma levels of PRL reached the maximal values within 30 min and then declined gradually in spite of continued TRH stimulation. Sulpiride given orally for 12 consecutive days in the luteal phase of the menstrual cycle caused a significant increase in the plasma PRL level. This increase was higher than that obtained in the experiment during the follicular phase. No significant change in the plasma level of 17 alpha OH delta 5-P was observed TRH sulpiride administration. In 3 out of 9 cases, plasma DHEA-S levels were elevated significantly on day 13 to 14 of the experimental course. These results indicate that the responsiveness of plasma levels of adrenal androgens to the raised plasma PRL level requires a certain period of time in the hyperprolactinemic state in the human female.

1. The membrane of Rhodospirillum rubrum chromatophores was disintegrated with mild detergents (cholate and deoxycholate) in order to study the spatial arrangement of the functional proteins in the photochemical apparatus and the electron transport system in the membrane. 2. The components solubilized from the membrane by a mixture of cholate and deoxycholate (C-DOC) were separated into four fractions by molecular-sieve chromatography in the presence of C-DOC; they were designated as F1, F2, F3, and F4 in the order of elution. The fractions were further purified by repeated molecular-sieve chromatography in the presence of C-DOC until each fraction was chromatographically homogeneous. 3. F1 appeared to be conjugated forms of F2. 4. The purified F2 was composed of a rigid complex having a weight of 7 X 10(5) daltons, containing approximately 10 different kinds of protein species with molecular weights of 3.8 X 10(4), 3.6 X 10(4), 3.5 X 10(4), 2.8 X 10(4), 2.7 X 10(4), 2.6 X 10(4), 1.3 X 10(4), 1.2 X 10(4), 1.1 X 10(4), and 1.0 X 10(4). The complex contained 33 bacteriochlorophylls, 4 iron atoms, and 90 phosphates, but no cytochrome, ubiquinone, or phospholipid. It showed the same reaction center activity as chromatophores, indicating that the complex was a unit of the photochemical apparatus (photoreaction unit). Each chromatophore of average size was estimated to possess about 24 photoreaction units. 5. The purified F3 showed an absorbance spectrum characteristic of reaction centers, and contained 3.4 bacteriochlorophylls, 2.0 bacteriopheophytins, and 1.9 acid-labile iron atoms, but no cytochrome or ubiquinone (C-DOC reaction center). It had a weight of 1.2 X 10(5) daltons, and the main components were 4 protein species with molecular weights of 2.8 X 10(4), 2.7 X 10(4), 2.6 X 10(4), and 1.0 X 10(4). 6. The purified F4 showed a molecular weight of about 11,000, and contained one mole of ubiquinone-10 per mole (ubiquinone-10 protein). 7. The reaction center activity of C-DOC reaction centers was stimulated by ubiquinone-10 protein. In addition, the reaction center oxidized reduced cytochrome c2 in the light, provided that ubiquinone-10 protein was present (photo-oxidase activity).

Adjuvant activity of chitin derivatives was examined in guinea-pigs and mice. Among derivatives of chitin tested, 30 and 70% deacetylated chitin (DAC-30 and DAC-70), were active as adjuvants for the circulating-antibody formation to bacterial alpha-amylase and for the induction of delayed-type hypersensitivity to azobenzenearsonate-N-acetyl-L-tyrosine. DAC-70 enhanced the helper T cell function, the generation of alloreactive cytotoxic T lymphocytes, and the activity of natural killer cells in mice, however, it was inactive as mitogen. Other derivatives of chitin showed weaker or no adjuvant activity compared with DAC-70. Carboxymethyl-, hydroxyethyl and dihydroxypropyl-chitin showed weak mitogenic activities on normal spleen cells.

Distribution of an androgen-dependent, 20,000-dalton nonhistone protein with a pI of about 11.5 (20K-NHP) was examined by electrophoretic techniques. Nuclei of the brain, liver, spleen, skeletal muscle, lung, and thymus of rats contained a negligible amount of 20K-NHP, whereas 20K-NHP was distinctly detectable, in different relative amounts, in the nuclei of the male accessory sex organs, with the dorsolateral prostate having the highest relative content (100%), followed by the coagulating gland (approximately equal to 16%), the ventral prostate (approximately equal to 6%), and the seminal vesicle (approximately equal to 2%). There were heterogeneous distributions of cytosol components, acid phosphatase isozymes, and nonhistone proteins in the dorsolateral prostate. Zinc was localized in the lateral lobe, and fructose and glucose were in the dorsal lobe. Cytosol proteins with pI 7.5, 8.2, and 8.5 were abundant in the dorsal lobe, and proteins with pI 7.4 and 8.0 in the lateral lobe. Acid phosphatase isozymes with pI 7.1, 7.4, 7.7, and 8.0 were abundantly distributed in the lateral lobe. Of the nonhistone proteins, 20K-NHP showed the highest content both in the lateral lobe and in the dorsal lobe. It was found that 20K-NHP was more abundantly distributed in the lateral lobe (maximally four times higher) than in the dorsal lobe. The heterogeneous distribution of 20K-NHP in the dorsolateral prostate was strikingly similar to that of zinc. It appears, therefore, that 20K-NHP is closely related to dorsolateral prostate zinc content.

Certain local tissue factors, such as growth factor, in addition to androgens, are involved in the prostate growth. The prostate contains two types of growth factors capable of stimulating DNA synthesis in BALB/3T3 cells. They were divided into low affinity (LoA) type and high affinity (HiA) type by a different affinity for heparin-Sepharose. HiA-type growth factor is further classified into acidic HiA and basic HiA types. Acidic HiA type could be purified from the Dunning tumor (R 3327), a rat prostatic adenocarcinoma, and has a molecular weight of about 19,000 and a pI of about 3.8. Basic HiA type could be isolated from the tissues of human benign prostatic hypertrophy and has a molecular weight of about 12,000 and a pI of about 10.5. They are inactivated by heat and acid treatments. Acidic HiA type appears to be involved in growth of the rat prostate epithelium, and LoA type growth factor is possibly relevant to reproductive physiology because of its coexistence with "probasin," a major secretory protein in the dorsolateral prostate having a strong affinity for spermatozoa.

In our previous study, we reported that pregnant blood was hypercoagulable and hypo-fibrinolytic. However, localized fibrinolytic activity, e.g. in the placenta, seemed to be present, because of increased FDP and fibrinopeptide B beta 15-42 during pregnancy. In this study, we tried to find out the changes in the coagulation-fibrinolysis system as well as cold insoluble globulin when stress such as operative procedure is being added. In 25 cases of cesarean section in full term pregnancy without onset of labor, blood samples were collected before operation, during operation and 3 hours, 6 hours, 12 hours, 24 hours and 48 hours post-operatively for estimation of the coagulation factors (FXII, IX, VIII, VII, X, V, II. XIII. Fbg), fibrinolytic factors (plg-Act, plg, SFMC, FDP), prekallikrein, plasma serin protease inhibitors (AT-III, alpha 2-PI, alpha 2-M, alpha 1-AT) and cold insoluble globulin. 30 normal term-pregnant women as well as 30 non-pregnant women were studied as the control. The result showed that the activities of both the intrinsic and extrinsic coagulation factors were markedly increased while the Fbg and AT-III were markedly decreased from during operation to 3 hours after operation. FXIII was markedly decreased and FII was slightly decreased from during operation. The marked decreases in prekallikrein, plg, alpha 2-PI, alpha 2-M, alpha 1-AT together and marked increases in plg-Act and FDP were noted from 3 to 12 hours after operation. A marked decrease in CIG was demonstrated immediately after the operation and continued until 48 hours after operation. These findings suggested that hypercoagulable state and secondary hyperfibrinolysis were associated during and after cesarean section. Decreases in Factor XIII and CIG seemed to be a general reaction in the mechanism of wound healing.

The rat prostate contains two types of growth factors capable of stimulating DNA synthesis in BALB/3T3 cells. These rat prostatic growth factors (RPGF) were separable by a different affinity for heparin: low affinity type RPGF and high affinity (HiA) type RPGF. About 80% of the RPGF in the cytosol from normal prostates was low affinity type, whereas more than 80% in the cytosol from the Dunning tumors was HiA type. Elution profile of HiA-RPGF showed two peaks of activity eluted from the heparin-Sepharose column, one at 1.3-1.4 M NaCl (HiA1-RPGF) and the other at 1.6-1.7 M NaCl (HiA2-RPGF). HiA2-RPGF could be purified 1100-fold from the Dunning tumor (AT-3 subline) in about 20% recovery by heparin-Sepharose chromatography. The partially purified HiA2-RPGF in the Dunning tumor has a molecular weight of about 19,000 and isoelectric point of about 3.8, and stimulated DNA synthesis at a concentration of about 0.25 nM. The activity was lost by heat treatment at 70 degrees C for 5 min and by acid treatment, whereas it was stimulated by incubating with dithiothreitol. The HiA2-RPGF did not have transforming growth factor activity at a concentration of 250 ng/ml or lower in the presence of epidermal growth factor.

Effects of oral and vaginal administration of LH-RH and [D-ALa-6, DesGly-10-NH2i1-LH-RH ethylamide (D-Ala-6-LH-RH-EA) on serum LH levels in ovariectomized, estrogen, progesterone treated rats were investigated. Oral administration of synthetic LH-RH induced a quick rise of serum LH levels with the greatest elevation at 15 min at any dose levels tested. On the other hand, oral administration of D-Ala-6-LH-RH-EA resulted in a slow but progressive rise of LH during 120 min of observation. The total amount of LH released by 10 mug of the analog was much greater than the total released by 1000 mug of LH-RH. Vaginal administration of 100 mug of LH-RH mixed with Carbowax induced a progressive rise of LHwhich was indistinguishable from that following 10 mug of the analogue, suggesting that the potency of the analogue is 10 times greater than that of LH-RH for vaginal administration. Ten mug of LH-RH given through the vagina induced a rapid rise of LH with the peak at 15 min, whereas 1 mug of the analog induced a slow but progressive rise. Greater resistance of D-Ala-6-LH-RH-EA than LH-RH against in vivo breakdown is postulated as one of the causes of greater and prolonged LH release by the former.

Mechanically isolated epithelium and stroma from benign hypertrophic prostates were highly pure on the basis of histochemical and biochemical criteria. By electrophoretic analyses, whole cellular and nuclear proteins were compared among whole tissues, epithelium, and stroma. The characteristic protein profiles of benign hypertrophic prostates were reflected in the electrophoretic patterns of the stroma. Two-dimensional gel electrophoretic patterns of the epithelium were different from those of the stroma with exception of about 18 major spots that were common to both fractions. Of the protein species separated, 35K/6.7 (molecular weight/pI) and 36K, which was composed of two species with pI of 4.4 and 4.6, were abundant in the epithelium and stroma, respectively. Nuclei prepared from whole tissues of benign hypertrophic prostates contained three kinds of nonhistone proteins (NHP) closely associated with benign prostatic hypertrophy (BPH); 42 K-NHP, 55 K-NHP and 190 K-NHP. Electrophoretic analysis of the nuclear proteins revealed that all the BPH-associated nonhistone proteins were abundantly distributed in the nuclei of the stroma.

Since Arimura et al (1975) reported the radioimmunoassay for somatostatin (GIF), the concentration of GIF in various organ and brain regions were determined by radio-immunoassay. Dubois et al (1975) reported that immunohistochemically somatostatin was located in the discrete cells of the pancreas as well as the hypothalamus, and from this result, they presented the concept of local hormone instead of systemic hormone which was up to that time accepted in endocrinology. In this study, we developed the high specific anti-GIF serum using rabbits, and with the micro immunodiffusion method, we demonstrated that the precipitin band formed a circular fusion between the GIF and anti-GIF serum. This pattern of reaction was also seen in decidual immunodiffusion. In addition, we developed the radioimmunoassay for GIF using this anti-serum and measured immunoreactive GIF-like substances in villi and decidua of early pregnancy. The concentration of GIF-like substances with 2 N acetic acid extracted of villi and decidua were 0 to 30 pg/0.1 g dry weight. At the same time, we demonstrated the presence of GIF-like substance-containing cells in the villi and decidua by indirect immunofluorescent method. The intensity of immunofluorescence was in cytotrophoblast rather than syncytiotrophoblast, and decidual stromal cell also reacted to the immunofluorescence.

The effect of the administration of sheep anti-LHRH gamma globulin (anti-LHRH-G) on implantation of fertilized ova was investigated in rats. Daily injections of 1 ml of anti-LHRH-G from Days 1 through 7 of pregnancy uniformly inhibited implantation of fertilized ova on Day 8, but viable sites, though considerably smaller in size than in control rats, became distinguishable on Day 14 in most rats. In some of these rats resorption of fetuses occurred, and others delivered pups 7-8 days after term. When the rats were given anti-LHRH-G from Days 3 through 5, the implantation was delayed by 5 days, but the gestation was not terminated. A single injection of 1 ml of anti-LHRH-G on Day 4 inhibited implantation on Day 8, but injection on Day 3 or 5 did not. The delayed implantation by anti-LHRH-G injected on Day 4 was nullified by concomitant administration of 2 sc injections of 1 mug of LHRH, or a single dose of 1 mug of estradiol. The data indicate that the hypothalamic LHRH is essentail on Day 4 of pregnancy for timely implantation of fertilized ova, probably by maintaining LH and, consequently, estrogen secretion.

A unique polypeptide containing the repeated structure of core sequence from cell adhesion molecules, poly(Arg-Gly-Asp), was successfully prepared by the polymerization procedure with diphenylphosphoryl azide. This polypeptide dramatically inhibited the aggregation of platelets induced by adenosine diphosphate (ADP) or malignant melanoma cells.

Structure-function studies for the antimetastatic activity of chemically modified chitin heparinoids composed of N-acetyl glucosamine units were performed in an experimental lung metastasis model. 6-O-Sulfated chitin (S-chitin) significantly inhibited the lung tumor colonization in proportion to the degree of sulfation. However, 6-O- and N-sulfated but partially N-deacetylated chitin (S-chitosan), and 6-O-carboxymethylated chitin (CM-chitin) had no effect. 6-O-Sulfated CM-chitin (SCM-chitin), which exhibited fairly low levels of anticoagulant activity, was also more effective than intact heparin. Furthermore, SCM-chitin with a high degree of sulfation (SCM-chitin III) caused a marked decrease of the number of lung tumor colonies in the spontaneous lung metastasis model. These results strongly suggest that 6-O-sulfate and N-acetyl groups in the glucosamine unit were required for the anti-metastatic effect of chitin heparinoids as well as heparin, and SCM-chitin III may be of therapeutic benefit for the prevention of tumor metastasis.

Induction of a specific antibody for methamphetamine, an antihypnotic drug, has been studied using carboxymethyl-chitin (CM-chitin) as a hapten carrier. The hapten-specific antiserum was induced by only a couple of hypodermal injections every 2 weeks. Inhibition of antigen-antibody complex formation was linearly related to methamphetamine concentration in the range 0.5 to 50 ng/50 microliter when an enzyme-linked immunosorbent assay was performed using an avidin-biotin-peroxidase complex. Little antibody directed against CM-chitin and CM-chitin oligomer was detected. Thus it seems to be advantageous in the hapten-bovine serum albumin system. The specificity of the antibody was high as shown by the use of various methamphetamine analogs.

The effect of multi-porous beads prepared from 80% deacetylated chitin on the activation of mouse peritoneal macrophages was examined. Deacetylated chitin bead (DAC-bead) preparations were shown to activate macrophages for tumoricidal activity depending on the increasing concentration of acetic acid used for the pretreatment of beads. The large DAC-bead was more susceptible to treatment with acetic acid than small DAC-bead, and showed more potent capacity for the activation of macrophages under the same pretreatment conditions with acetic acid. Deacetylated bead preparations, on the other hand, showed less activities. In addition, DAC-bead pretreated with acetic acid stimulated macrophages to produce interleukin 1. The possibilities of multi-porous beads as cancer chemotherapeutic-carrier were examined by the method of column chromatography and of in vitro antitumor experiment. Forty-four percent of adriamycin adsorbed on the surface of and in bead was released within the first 60 min. of elution, and then adriamycin was released more slowly in proportion to the elution time. Antitumor activity of adriamycin-adsorbed bead was less effective than that of free adriamycin if they were compared on the basis of total content of adriamycin.

Women, most of whom had regular menstrual cycles, were administered D-Leu6,des-Gly10-NH2)-luteinizing hormone-releasing hormone (LH-RH) ethylamide (D-Leu6-LH-RH-EA)via different routes during the early or midfollicular phase of the cycle. Plasma LH, follicle-stimulating hormone (FSH), and estrogen levels were determined by radioimmunoassay before and after administration of D-Leu6-LH-RH-EA. plasma LH and FSH increased and reached peak levels 3 to 4 hours and 3 to 6 hours, respectively, after subcutaneous injection of 25 mug of the analog of LH-RH. Intravaginal or intrarectal application of 2 mg of D-Leu6-LH-RH-EA also increased plasma LH and FSH levels in most of the women, but the magnitude of the rise, the time of initiation of response, and the peak level varied among the women. The plasma estrogen level also rose after administration via either route.

The substrate specificity in the hydrolysis of L-, DL-, and D-BAPA (benzoylarginine-p-nitro-anilide) by copoly (L-Cys, L-Glu) and copoly (D-Cys, D-Glu) was studied, and enzyme-like stereospecific hydrolyses by poly-alpha-amino acids were identified for the first time. The L-type copolymer hydrolyzed L-BAPA faster than D-BAPA and the rates (v) of BAPA hydrolyses by L-type copolymer were found to be in the order vL greater than vDL greater than vD. On the other hand, the D-type copolymer hydrolysed D-BAPA faster than L-BAPA and the rates of BAPA hydrolyses by D-type copolymer were in the order vD greater than vDL greater than vL. In all cases, the reaction followed Michaelis-Menten kinetics when the substrate concentration was corrected, and the optimum conditions of the reaction were pH 6.0 and 40 degrees. The activity appeared after a certain amount of BAPA had combined with the polymer. D- and L-substrates combine competitively with the polymer and the different rates of hydrolysis are presumably due to the different substrate configurations in relation to the conformation of the active site in the polymer. The polymer shows activity near the range of random coil conformation, where some alpha-helical conformation is still present. Only some of the cysteine residues in the copolymer are involved in the hydrolytic activity.

We have investigated the inhibitory effect on experimental or spontaneous lung metastases of polypeptides which contain repetitive structures of the Arg-Gly-Asp (RGD) or Tyr-Ile-Gly-Ser-Arg (YIGSR) sequence derived from adhesion molecules, and studied their biological characterisation after administration. In the spontaneous metastasis model, multiple intravenous (i.v.) administrations of poly (RGD) and poly (YIGSR) resulted in a reduction of lung tumour colonies, although the monomer peptides, RGD or YIGSR, had no effect under these conditions. The treatment with poly(RGD) substantially prolonged the survival time for mice injected i.v. with B16-BL6 cells as compared to the treatment with RGD and random poly(R, G, D). Tumour cell adhesion to the fibronectin-substrates was remarkably inhibited by adding poly(RGD) freely in solution. Poly(RGD) was found to inhibit completely the ability of platelets to enhance tumour cell adhesion to fibronectin-substrate and tumour cell-elicited platelet aggregation in vitro, but poly(R, G, D) had no such effect. We also found that poly(RGD) led to a decrease in the arrest and retention of tumour cells after its co-injection with radiolabelled tumour cells and that the radiolabelled polypeptide can be at least decomposed into small fragments during circulation. Poly(RGD) was found to be still active in inhibiting experimental lung metastasis even when the contributions of NK cells or macrophages were removed from this system after pretreatment with anti-asialo GM1 serum, 2-chloroadenosine or carrageenan. The results indicate that the poly(RGD)-mediated inhibition of tumour metastasis may be due to the interference of the adhesive interaction of tumour cells with a specific site in the target organs. Derivatives of polypeptides which contain RGD and/or YIGSR sequences derived from cell adhesion proteins may thus provide a promising approach for the control and prevention of cancer metastasis.

The heptapeptide Ile-Arg-Ile-Cys-Arg-Lys-Gly-ethyl ester, having the amino acid sequence around the SH1 of myosin heavy chain, was coprecipitated with F-actin after ultracentrifugation. The heptapeptide inhibited the formation of acto-S-1 rigor complex by competing with S-1 for actin. Assuming a simple competitive inhibition, the dissociation constant of acto-heptapeptide complex was evaluated as 0.23 mM. An N-terminal tripeptide derivative from the heptapeptide Ile-Arg-Ile-methyl ester also formed a complex with F-actin with a dissociation constant of 1.1 mM. However, the other piece, Cys-Arg-Lys-Gly-ethyl ester, and a tetrapeptide, Val-Leu-Glu-Gly-ethyl ester, having the sequence adjacent to the N-terminal of the heptapeptide, scarcely bound with F-actin. These results suggest that part of the actin-binding site of myosin heavy chain around SH1 (Katoh, T., Katoh, H., and Morita, F. (1985) J. Biol. Chem. 260, 6723-6727) has the sequence of Ile-Arg-Ile and it is located adjacent to SH1 on its N-terminal side.

The purpose of this study is to investigate the ovulatory effect of the combined therapy with bromocriptine (Brc), clomiphene (Cl) and HMG. We investigated 25 patients with polycystic ovary syndrome (PCOS) who had anovulatory normoprolactinemia and failed to respond to Cl therapy alone. The results were as follows: 1) The ovulation rate with Method I(Brc/Cl) was 64.0% (16/25) in 25 cases, and 59.8% in 122 cycles. 2) Resting levels of serum dehydroepiandrosterone sulfate in the effective group with Method I were significantly lower than those in the non-effective group. 3) With Method I, serum prolactin (PRL), LH and testosterone were significantly decreased, while estradiol and progesterone concentrations were significantly increased in the effective group. However, there were no significant changes in the hormone levels except PRL in the non-effective group. 4) Out of the 9 nonrespondent Method I cases, 5 additional cases ovulated with both Method II(Brc/Cl+ a small dose of HMG) and Method III(Brc/Cl+ an increased dose of HMG-HCG). The total ovulatory rate was 84.0% (21/25) in 25 cases and 59.4% (85/143) in 143 cycles. 5) The total pregnancy rate was 43.8% (7/16). Six cases were normal pregnancies, one case was a twin pregnancy and there was no case of abortion. Our results suggested that the combined therapy with Brc, Cl and HMG had almost the same therapeutic effect as the HMG-HCG therapy in severe cases of PCOS.

Anti-cell adhesive activity was examined by the synthetic polypeptide, containing repetitive Arg-Gly-Asp sequence of cell attachment site from fibronectin, poly (Arg-Gly-Asp). The attachment of tumour cells to fibronectin substrate was specifically inhibited by adding poly (Arg-Gly-Asp) in cell surface receptor-mediated and divalent cation-dependent manners, but not by unrelated peptides. In our previous study, the lung metastatic formation of tumour cells was dramatically reduced by intravenous co-injection of anti-cell adhesive poly (Arg-Gly-Asp) with B16-BL6 melanoma cells. These findings suggest that polypeptide-mediated inhibition of pulmonary metastasis is partly due to interference with tumour cell adhesion to the substrates including fibronectin in target organs or tissues.

Growth hormone release-inhibiting hormone (somatostatin), a hypothalamic peptide that inhibits the release of growth hormone and also the secretion of insulin glucagon, and gastrin, was found in the rat stomach and pancreas in a concentration similar to that in the hypothalamus, as measured by radioimmunoassay. Somatostatin was also found in the duodenum and jejunum, but in a smaller concentration. Gel filtration of the extracts of the pancreas and stomach on Sephadex G-25 yielded two immunoreactive peaks, one corresponding in each case to the somatostatin tetradecapeptide. The hormone was not detected in other viscera or the ovaries. The results imply that somatostatin may be synthesized in the pancreas and the stomach in addition to the brain, and may be involved in local regulatory mechanisms for pancreatic and gastric secretion as well as secretion of growth hormone.

The purpose of this study was to investigate the correlation between gonadotropin (Gn) and prolactin (PRL) releasing activities on the hypothalamo-pituitary axis in the luteal phase of the normal menstrual cycle and in the early gestational period. The plasma levels of LH-beta subunit (LH-beta) following an intravenous bolus of 100 micrograms LHRH did not change significantly in early pregnancy. The plasma PRL levels, as well as the Gn levels, were significantly elevated (p less than 0.05) after the administration of 100 micrograms LHRH in the luteal phase, but did not rise in early pregnancy. The intravenous bolus of 10mg metoclopramide (MCP) had no effect on plasma LH-beta, LH, FSH, estradiol or progesterone in the luteal phase or early pregnancy. The plasma PRL levels remained significantly (p less than 0.001) elevated after the administration of MCP. However, there was no significant difference between PRL response to MCP in the early pregnant and luteal phase groups. In the luteal phase, we could show the correlation between Gn and PRL releasing mechanisms on hypothalamo-pituitary axis, which could not be found in early pregnancy.

It is very common for intraarterial infusion therapy of some anticancer agent to be effective against hepatocellular carcinoma. In this case, the patient was a 74-year-old man who suffered from very advanced hepatocellular carcinoma with tumor thrombus of the intrahepatic portal vein and IVC. He was treated with intraarterial infusion of CDDP, Etoposide, 5-FU, through a catheter placed in the proper hepatic artery. CDDP (30 mg/day) and Etoposide (60 mg/day) were given once every 5 days, and then 5-FU(250 mg/day) was infused daily for 26 days. The patient underwent this protocol study twice in 3 months. After the intraarterial infusion, transarterial embolization using CDDP (100 mg) powder added to lipiodol and aluminum stearate as suspension was done a month later. The tumor regression rate was 84% after intraarterial infusion of CDDP, Etoposide and 5-FU. The tumor thrombus in the intrahepatic portal vein and IVC had completely disappeared. We could not find lipiodol accumulated in the tumor after TAE. Thus, we assumed that the remaining tumor was a necrotic scar and that a complete response was obtained in the patient. There were some side effects, such as nausea, vomiting, pancytopenia and gastritis but no severe complication occurred.

A new arginine derivative, N-benzyloxycarbonyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide hydrochloride (ZPVAPA.HCl) was synthesized by the condensation of N-benzyloxy-carbonyl-L-phenylalanyl-L-valine and L-arginine-p-nitroanilide dihydrochloride using dicyclohexylcarbodiimide as a coupling reagent and 1-hydroxy-benzotriazole as an additive. L-ZPVAPA.HCl was split by trypsin more readily than Na-benzyloxycarbonyl-L-arginine-p-nitroanilide hydrochloride (L-ZAPA, HCl), Na-benzoyl-L-arginine-p-nitroanilide hydrochloride (L-BAPA.HCl), Na-tosyl-L-arginine-p-nitroanilide hdyrochloride (L-TAPA.HCl) and Na-benzoyl-DL-arginine-p-nitroanilide hydrochloride (DL-BAPA.HCl) by factors of 100, 400, 600, and 1,200, respectively. Low concentrations of dimethyl formamide (DMF) enhanced the trypsin-catalyzed hydrolyses of L-ZAPA.HCl and L-TAPA.HCl, contrary to the findings of other authors that DMF has no effect on the tryptic hydrolysis.

Four analogues of LH-releasing hormone (LH-RH) were injected iv. in normal men at a dose of 25 mug: des-Gly-10NH2-LH-RH ethylamide (9-EA-LH-RH); des-Gly-10-NH2-LH-RH propylamide (9-PA-LH-PH), d-ala-6-LH-RH; and D-Ala-6, des Gly10-NH2-LH-RH ethylamide (D-Ala-6,9-EA-LH-RH). Each peptide tested was found to be more potent than LH-RH in releasing LH and, to a lesser extent, FSH. The times of the peak values of LH after the analogues were similar to LH-RH (about 20 minutes). However, LH as well as FSH levels were considerably delayed in returning to baseline after administration of D-Ala-6,9-EA-LH-RH. The half-time disappearances of the 9-EA and 9-PA analogues could be tested and were found to be similar to that of LH-RH. The increased potency and prolonged duration of action of D-Ala-6,9-EA-LH-RH suggest the potential usefulness of this compound in stimulating ovulation and perhaps spermatogenesis.

A growth factor capable of stimulating DNA synthesis of BALB/3T3 cells was purified about 1,000-fold from the cytosol of human benign hypertrophic prostates by heparin-Sepharose chromatography; the growth factor bound to the column in the presence of 0.5 M NaCl was eluted with 1.5-1.7 M NaCl. Its molecular weight and isoelectric point were estimated to be 11,000-13,000 and 10.5, respectively. It was sensitive to heat- and acid-treatments but resistant to disulfide-reducing agent. The final preparation was able to stimulate DNA synthesis at 10 ng/ml. The degree of stimulation was dependent on serum concentration in the assay system; the degree of maximum stimulation increased about 5 times as serum concentration increased from 0.2 to 2%.

Nuclei of the dorsolateral prostate of rats contain a large amount of androgen-dependent non-histone protein (20K-NHP) (mol. wt. not equal to 20,000; pI not equal to 11.5) (Matuo et al. (1]. Its content in the nuclei increased most markedly during 4-8 weeks of age, when functional differentiation of the prostate was most active on the basis of the changes of major cytosol proteins and zinc. Nuclei of the Dunning tumors originating in the dorsolateral prostate were found to lack 20K-NHP regardless of androgen dependency, indicating the disappearance of the 20K-NHP from the nuclei by dedifferentiation. These suggest that the 20K-NHP is an important nuclear protein for differentiation of the dorsolateral prostate cells.

A series of C-terminally fluorinated analogs of luteinizing hormone-releasing hormone (LH-RH) was synthesized by a combination of solid-phase and classical techniques and found to have interesting properties when assayed for LH- and FSH-releasing activities over a prolonged period of time in immature male rats. One of the peptides, desGly-10-LH-RH-2,2,2-trifluoroethylamide, was found to be about nine times more effective than LH-RH in releasing LH, or approximately twice as active as the corresponding alkylamide peptide, desGly-10-LH-RH-ethylamide. However, desGly-10-LH-RH-2,2,3,3,3-pentafluoropropylamide was only slightly more active than LH-RH and considerably less active than the corresponding propylamide analog. A family of peptides was also prepared containing D-alanine in position six of the chain in conjunction with the C-terminal modifications. D-Ala-6,desGly-10-LH-RH-2,2,2-trifluoroethylamide, surprisingly, gave patterns of gonadotropin release which were only as intense and virtually identical with those obtained with D-Ala-6,desGly-10-LH-RH-ethylamide which in turn releases about 20 times more LH than a similar dose of LH-RH. D-Ala-6,desGly-10-LH-RH-propylamide also gave almost identical patterns of gonadotropin release. D-Ala-6,desGly-10,LH-RH-2,2,3,3,3-pentafluoropropylamide was considerably less potent, being only five times more effective than LH-RH.

The potential usefulness of a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHPAS), in the stabilization of acidic and basic fibroblast growth factors (FGFs) was examined. Among several detergents, CHAPS was found to be not only non-cytotoxic but also most useful in handling the diluted preparations of FGFs. The advantages are as follows: 1) at lower concentrations than 0.01% CHAPS did not affect growth factor activity of calf serum (CS) and the growth rate of BALB/c 3T3 cells. The primary culture of rat prostate epithelium and colony formation of NRK-49F cells were hardly influenced by CHAPS lower than 0.003%; 2) the loss of FGFs that usually occurs due to their adherence to the surface of storage containers was effectively prevented by inclusion of 0.1% CHAPS; 3) the recovery of FGFs after storage or dialysis was significantly enhanced by inclusion of 0.1% CHAPS; 4) CHAPS at lower concentrations than 0.1% does not interfere with amino acid analysis, except that Thr may be misled only when the ratio of protein/CHAPS is low; 5) amino acid sequence analysis was hardly disturbed by CHAPS up to 0.5%. These results indicate that CHAPS is useful as a stabilizing agent for various kinds of polypeptides capable of showing biological activity at a low concentration.

Rat prostate extracts contain an abundant 20-22 kilodalton heparin-binding protein with near identical chromatographic properties, but only 0.2-1% of the mitogenic activity, of bovine brain heparin-binding growth factor-1 (acidic fibroblast growth factor). Amino terminal amino acid sequence (met-met-thr-asp-lys-asn-leu-lys-lys-lys-ile-glu-gly-asn-trp-arg-thr-val -tyr- leu-ala-ala-ser-?-val-glu-lys-ile-asn-glu-gly-ser-pro) and immunochemical analysis revealed that the protein is identical to the androgen-dependent protein "probasin".

Proteins in the cytosol, postnuclear particulate, and nuclear fractions from seven specimens of normal prostate from bladder cancer patients, 14 specimens of benign hypertrophic prostate (BPH), and three specimens of cancerous prostate were analyzed and compared by SDS-polyacrylamide slab gel electrophoresis. Abundant protein species in the cytosol fractions were 60K (species having a molecular weight of about 60,000) and 42K; their relative contents were about 35% for 60K and about 12% for 42K. In the postnuclear particulate fraction, 42K was the most abundant (about 10% of the total). The contents of these major protein species were similar in specimens of normal and diseased prostates. In addition, there are marked similarities in the electrophoretic patterns for all the protein (24-29 species) in the cytosol and postnuclear particulate fractions of the human prostate, except for four minor species in the cytosol fraction. Of the nuclear proteins, the content of core histones (H2A, H2B, H3, and H4) was fundamentally similar among all the specimens, whereas the content of H1 histone was different from one specimen to another. The most remarkable and significant difference was that the 42K-NHP (nonhistone protein having a molecular weight of about 42,000), 55K-NHP, and 190K-NHP concentrations were significantly higher in BPH than in normal and cancerous prostates.

Growth factors, as detected by DNA synthesis stimulating activity for BALB/c 3T3 cells, in normal, benign hypertrophic and cancerous human prostates were analyzed. The total content (units per gram of tissue) in benign hypertrophic prostate was two to four times higher than those in normal and cancerous prostates. In all the three groups, heparin-binding growth factor, growth factor adsorbed to heparin-Sepharose in the presence of 0.5 NaCl. accounted for about 80-95% of the total growth factor content. Qualitative analysis using a heparin-Sepharose column revealed two types of heparin-binding growth factor in the prostates, one eluted from the column at 1.2-1.3 M NaCl and the other at 1.5-1.8 M NaCl. The latter was the predominant type in all groups. In addition to the growth factors detected with BALB/c 3T3, a growth factor with specific action upon MC3T3-E1 mouse osteoblasts was found in prostatic cancer, but not in normal and benign hypertrophic prostates.

The effect of the administration of sheep anti-LHRH gamma-globulin (anti-LHRHG) on the maintenance of pregnancy was investigated in rats. Nidation was confirmed by laparotomy on day 7 or 8 of pregnancy. In one experiment, rats were then injected iv with 1 ml anti-LHRHG or normal sheep gamma-globulin (NSG) daily from days 7 to 11. The uteri were inspected again on day 14 of pregnancy, when it was found that complete resorption of the fetuses had occurred in the anti-LHRHG-treated rats, but that the fetuses were normal in the NSG-treated control rats. The effect of a single injection of 1 ml of anti-LHRHG on day 7, 8, 9, 10, 11, or 12 of pregnancy was also investigated. Administration on day 9 or 10 resulted in complete resorption of the fetuses by the time of the 2nd inspection on day 14, and treatment on day 8 or 11 was partially effective. However, treatment on day 7 or 12 exerted little effect on viability of the fetuses. None of the rats showed vaginal bleeding following treatment with anti-LHRHG. Termination of pregnancy by anti-LHRHG could be prevented by SC injection of 1 mug LHRH twice daily, or by 4 progesterone SC once daily, from days 9 through 12 of pregnancy. The ovaries of the rats treated with anti-LHRHG from days 7 to 11 were smaller than those of the NSG-treated control rats, and some of the corpora lutea underwent cystic degenerative changes. Lutein cells of the former were also smaller than those of the latter. Serum progesterone levels were reduced after a single injection of anti-LHRHG on day 9 or 10, but not on day 7 or 12 of pregnancy. There was excellent agreement between the reduction of serum progesterone and fetal resorption. Serum LH levels were low on days 7 through 12 in the anti-LHRHG-treated as well as the NSG-treated rats, and the possible suppressive effect of anti-LHRHG on LH could not be revealed because of insufficient sensitivity of the radioimmunoassay method. No significant difference was observed in serum prolactin levels between the groups of rats. The results clearly indicate that LHRH, by maintaining progesterone secretion, is indispensable on days 9 and 10 of pregnancy for the maintenance of pregnancy.

The purpose of this study is to investigate the combined effect of bromocriptine and clomiphene citrate (Cl) treatment on 35 patients with normoprolactinemic amenorrhea who failed to respond to Cl alone. Ovulation was restored by this treatment in 20 of these patients (57.1%), and the pregnancy rate was 26.7%. This treatment was effective in 11 of 15 women with polycystic ovary like syndrome (73.3%). Resting levels of prolactin, estradiol and testosterone in the effective group were significantly higher than those in the non-effective group. As a result of the treatment, the prolactin and LH levels were significantly decreased, and estradiol and progesterone levels were significantly increased in the effective group. Before the treatment, the responsiveness of LH to LHRH in the effective group was significantly higher, but the responsiveness of prolactin to TRH in this group was not significantly higher than that in the non-effective group. After the treatment, the LH-releasing response following a premarin injection in the effective group was significantly higher than that in the non-effective group. The present results indicate that the therapeutic effect of this treatment may be primarily due to the restoration and improvement of the impaired hypothalamo-pituitary system without the suppression of pituitary prolactin secretion.

The interactions of the "antidementia drug" pantoyl-gamma-aminobutyric acid (pantoyl-GABA) with gamma-aminobutyric acid (GABA) receptors were investigated by studies on bindings of radiolabelled ligands in rat brain. Pantoyl-GABA inhibited the binding of [3H]GABA to GABAA receptors and those of [3H]baclofen and [3H]GABA to GABAB receptors in the rat cerebral cortex. These data suggest that pantoyl-GABA interacts with both types of GABA-receptors in the rat brain.

Sixteen normal pregnant woman at delivery were administered 10 mg of Metoclopramide (MCP) intravenously, and the Prolactin (PRL) levels in the maternal plasma and in the amniotic fluid were measured by RIA before and after the administration, and in the umbilical plasma after the administration. Eight other pregnant women at term were studied similarly without the administration of MCP and served as the control. In this experiment, there was a significant increase only in the maternal plasma PRL but not in the amniotic fluid PRL nor in the umbilical plasma PRL. Furthermore, by using an intrauterine pressure catheter and a maternal intravenous cannula we measured the amniotic fluid PRL and the maternal plasma PRL about every 20 minutes during 3 hours before and after the intravenous administration of MCP 10mg. The maternal plasma PRL increased promptly and remained high for 150 minutes, while there was no significant change in the amniotic fluid PRL. This obvious discrepancy supports the hypothesis that decidua is the source of amniotic fluid PRL and suggests an independent regulation of amniotic fluid PRL.