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K J Chang

Publications and source records attributed to K J Chang.

At least 145 records · Page 8Linked to original sources
PubMed1993

DNA methylation is not involved in growth regulation of gene expression of proliferating cell nuclear antigen.

Expression of proliferating cell nuclear antigen (PCNA) gene is growth regulated. By Southern blot analysis with restriction enzymes such as HpaII and MspI, no change of DNA methylation state in this gene was detected during rat liver regeneration. There is only one HpaII site located in the promoter region of rat PCNA gene and this HpaII site was found to be demethylated in both normal and regenerating livers. Human hepatoma cell lines Hep G2 and Hep 3B were used to study the relation of PCNA gene expression and the DNA methylation. There are 16 HpaII sites in human PCNA gene, and according to the HpaII and MspI restriction patterns, many of the HpaII sites were methylated in vivo. Upon serum stimulation of quiescent cells, no change of DNA methylation state in the HpaII and HhaI sites was found. Demethylation by the methylation inhibitor, 5-azacytidine, did not affect the expression of PCNA gene in the hepatoma cells. With the human primary fibroblasts, Y2, the demethylation by 5-azacytidine did not seem to change the growth dependence of PCNA gene expression. This is consistent with the observation that the expression of PCNA gene of some cultured cell lines such as CHO.K1, in which the PCNA gene was unmethylated, showed growth dependence. Also, no variation in methylation pattern of PCNA gene was found among the different rat tissues in which the expression of PCNA varies. Therefore, we conclude that DNA methylation is not involved in growth regulation of the PCNA gene expression.

Animals↗
PubMed1993

Circulating intercellular adhesion molecule-1 and lymphocyte subsets involved in immune response of breast cancer.

To understand the immune responses of patients with breast cancer, circulating intercellular adhesion molecules-1 (ICAM-1) and lymphocyte subsets were measured in patients with breast cancer and in healthy persons. There were 10 patients of with metastasis, 11 patients breast cancer without metastasis and 9 age-and-sex matched healthy persons. Their serum samples were counted for circulating ICAM-1 levels, and blood cells were calculated for lymphocyte subpopulation including CD3, CD4, CD8 and CD19 by using flow cytometry. Higher levels of circulating ICAM-1 were noted in patients with breast cancer and metastasis than those without metastasis (p < 0.01). Lower percentage of CD8 lymphocyte subset were found ih patients with breast cancer and metastasis than those without metastasis (p < 0.05). Shedding of ICAM-1 antigens from the tumor may block the attachment of cytotoxic T cells, and decreased numbers of cytotoxic T cells may be unable to inhibit the spread of cancer cells. Immune responses of patients with breast cancer may be different between metastatic status and limited status. Further investigations should correlate the results of tissue stains of breast cancer with ICAM-1 and lymphocyte markers with those from peripheral blood.

Adult↗
PubMed1993

Liver resection for hepatic metastasis from colorectal carcinoma.

One hundred and eighteen patients who suffered from metastatic liver tumors from colorectal carcinoma during the period from January 1980 to June 1991 at the National Taiwan University Hospital were retrospectively reviewed. The modes of treatment included hepatic resection (n = 18), transarterial embolization (TAE, n = 17), chemotherapy only (C/T, n = 36), and no treatment (n = 47). In the 18 patients who underwent hepatic resection during the last 10 years, the types of surgery included: wedge resection (n = 8), segmentectomy (n = 2), right hepatic lobectomy (n = 5), left hepatic lobectomy (n = 2) and extended right hepatic lobectomy (n = 1). There were no operative mortalities in this series. The overall three-year actuarial survival rate of the resection group was 57.5%. It was better than that of the TAG group (17%, p < 0.05). For a solitary tumor, the three-year actuarial survival rate rose to 64% in the resection group. There were no three-year survivors in the nonsurgical solitary tumor group. This experience defends aggressive surgical treatment of resectable hepatic metastatic liver cancer from colorectal carcinoma as a safe procedure which is able to prolong a patient's life.

Adult↗
PubMed1993

[Seroepidemiological investigation on Japanese encephalitis in Taiwan (January 1989-December 1991)].

The hemagglutination inhibition (HI) test was used to detect sero-confirmed cases of clinically suspected Japanese encephalitis. The results revealed a total of 76 sero-confirmed cases; 24, 27 and 25 cases respectively for the year 1989, 1990 and 1991. The percentages of sero-confirmed cases with respect to reported cases were 12.97%, 12.33%, 10.04% respectively. The mean percentage was 11.64%. The mean number of sero-confirmed cases in recent three years was 25, and morbidity rate per 100,000 population was 0.125. The number of sero-confirmed cases in 1970 was 269, and morbidity rate per 100,000 was 1.86. The comparison of these two groups showed the ratio of 1:11 and 1:15. It demonstrated that the period of 50% sero-conversion of HI antibody positive pigs appeared two months earlier in the middle of May currently as against the middle of July during the years 1966-1968. The period of JE prevalence likewise shifted two months earlier to the months of May, June and July, with a peak in June in 1989-1991 as against the months of July, August and September with a peak in August in 1966-1968. There occurred a few sporadic cases in other months away from the period of major epidemic. With regard to the geographical distribution of the sero-confirmed cases in recent three years, most JE cases were detected in the Taipei basin and the Kaohsiung-Pingtung river basin. The next numerous cases were found in the terrace which extends from Taoyuan, Hsinchu and Miaoli to the central Taiwan and in the Chiayi-Tainan plain. Only four cases were reported from Ilan, Hualien and Taitung counties. During the years between 1955 and 1967, the vast majority of cases occurred among children, especially of 2-9 years of age. The next suffered group was the age group of 10-14 years. In the meanwhile, the current major group of victim was the age group of 10-14 years and its morbidity rate per 100,000 population was 0.30. The second suffered group was the age group of 20-24 years and its morbidity rate was 0.28, and the third was the age group of 0-9 years and its morbidity rate was 0.16. The percentages of sero-confirmed cases of the above each age group with respect to all sero-confirmed cases were 23.68%, 21.05%, 22.36% respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1993

BW373U86: a nonpeptidic delta-opioid agonist with novel receptor-G protein-mediated actions in rat brain membranes and neuroblastoma cells.

BW373U86 is a potent and highly selective nonpeptidic agonist for delta-opioid receptors. To determine its ability to couple with G protein-linked second messenger systems, this study examined the effects of BW373U86 on the inhibition of adenylyl cyclase and the stimulation of low-Km GTPase activity. In rat striatal membranes, BW373U86 inhibited basal adenylyl cyclase activity in a GTP-dependent manner, with maximal inhibition levels similar to those of the prototypic delta agonist [D-Ser2,Thr6]Leu-enkephalin (DSLET). However, BW373U86 was approximately 100 times more potent than DSLET in inhibiting adenylyl cyclase. Analysis of the inhibitory activity across 10 brain regions revealed that both low and high concentrations of BW373U86 inhibited adenylyl cyclase activity in a manner similar to that of DSLET. Inhibition of adenylyl cyclase by BW373U86 was delta receptor selective, because the delta receptor-selective antagonist naltrindole was significantly more potent than naloxone and the mu receptor-selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 was ineffective in blocking BW373U86 inhibition. BW373U86 also inhibited adenylyl cyclase activity in membranes prepared from NG108-15 cells, with an IC50 value 5 times lower than that of DSLET. This increased potency was not observed in concentration-effect curves for agonist-stimulated low-Km GTPase in NG108-15 membranes. BW373U86 is a competitive inhibitor of [3H]diprenorphine at delta receptors of NG108-15 cell membranes. However, unlike DSLET, BW373U86 displacement of [3H]diprenorphine binding to NG108-15 cell membranes was not affected by sodium and guanine nucleotides. This lack of GTP effect on binding apparently produced slow dissociation rates for this agonist, because naltrindole was less potent in blocking BW373U86 inhibition of adenylyl cyclase when membranes were preincubated with this agonist. These results demonstrate the novel finding that the binding of a full agonist to a G protein-coupled receptor is not regulated by GTP, and they also show how the lack of regulation in receptor binding affects agonist potency.

Adenylyl Cyclase Inhibitors↗
PubMed1993

A novel, potent and selective nonpeptidic delta opioid receptor agonist BW373U86.

Four different opioid receptor binding assays and three different isolated tissue studies were used to screen for delta receptor-selective nonpeptidic compounds. (+/-)-4-((alpha-R*)-alpha-((2S*,5R*)-4-Allyl-2,5- dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N,N-diethylbenzamide (BW373U86) was a potent delta receptor-selective ligand in receptor binding assays. The Ki values were 1.8 +/- 0.4, 15 +/- 3, 85 +/- 4 and 34 +/- 3 nM for delta, mu, epsilon and kappa receptor binding sites, respectively. BW373U86 inhibited electrically evoked muscle contraction of mouse vas deferens with an ED50 value of 0.2 +/- 0.06 nM. This inhibitory effect of BW373U86 was antagonized by the delta receptor-selective antagonist naltrindole in a competitive manner: the Schild plot indicated a slope of 1 and a pA2 value of 9.43 (Ke = 3.7 x 10(-10) M), which is consistent with the high affinity of naltrindole in delta receptors. BW373U86 did not interact significantly with other receptors. BW373U86 inhibited the acoustic startle reflex after subcutaneous administration from 0.2- to 2-mg/kg doses in rats, and this inhibition was blocked by naltrindole. BW373U86 also induced a dose-dependent increase of locomotor activity in rats at similar doses. This effect was inhibited by naltrindole. These data suggest that BW373U86 is a potent and selective nonpeptidic delta agonist, and it elicits distinct in vivo pharmacological activities.

Amino Acid Sequence↗
PubMed1993

Antinociceptive actions of BW373U86 in the mouse.

This study explored the antinociceptive properties of (+-)-4-[(alpha- R*)-alpha-[(2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinol]-3-hydroxy benzyl] - N,N-diethyl-benzamide dihydrochloride (BW373U86) a nonpeptidic compound proposed to be a delta opioid agonist, using three models of nociception and four routes of administration in mice. BW373U86 produced dose- and time-dependent, naloxone-sensitive antinociception in both the tail-flick and tail-pinch assays when given intrathecally (i.t.). However, at doses up to 187 nmol/mouse, i.c.v. BW373U86 was inactive in the tail-flick or tail-pinch assays. Additionally, at doses up to 187 mumol/kg, BW373U86 was not active after i.p. or p.o. administration in these endpoints. Further, in the tail-flick test, i.c.v. BW373U86 did not antagonize the antinociceptive effects of i.c.v. [D-Pen2,D-Pen5]enkephalin or [D-Ala2,Glu4]deltorphin, but partially antagonized the effects of i.c.v. morphine. In the acetic-acid abdominal constriction assay, BW373U86 produced dose-dependent antinociceptive effects when given by the i.p., i.c.v. or i.t., but not by the p.o., routes. Intrathecal BW373U86 was 663-fold more potent in the abdominal constriction assay than when given by the same route in the tail-flick test. The effects of an A70 dose of i.p. or i.c.v. BW373U86 in the abdominal constriction assay were partially antagonized by i.c.v. naloxone, but not by i.c.v. N,N-diallyl-Tyr-Aib- Aib-Phe-Leu-OH, where Aib is alpha-aminoisobutyric acid (ICI-174,864) or naltrindole. In contrast, i.t. naloxone, ICI-174,864 or naltrindole antagonized the antinociceptive effect of i.p. or i.t. BW373U86 in the abdominal constriction assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Abdomen↗
PubMed1993

Discriminative stimulus effects of BW373U86: a nonpeptide ligand with selectivity for delta opioid receptors.

Several opioid agonists were evaluated in pigeons trained to discriminate i.m. injections of sterile water from either the mu agonist morphine (5.6 mg/kg), the kappa agonist bremazocine (0.032mg/kg) or (+/-)-4-((alpha-R*)-alpha-((2S*,5R*)-4-allyl-2,5-dimethyl-1- piperazinyl)-3-hydroxybenzyl)-N,N-diethylbenzamide (BW373U86; 0.56 mg/kg). Pigeons were trained to peak one of two keys on a fixed-ratio 20 schedule for food reinforcement. The pattern of substitution of mu, kappa and delta selective agonists in the three groups of birds suggested that the discriminative stimulus effects of morphine, bremazocine and BW373U86 were different; however, a component of the discriminative stimulus effects of BW373U86 appeared to be shared with morphine. Apparent pA2 values for naltrexone with morphine, bremazocine and BW373U86 were 7.6, 6.8 and 6.3, respectively. The apparent pA2 value for naltrindole with BW373U86 was 8.3. Naltrindole (10.0 mg/kg) produced a 3-fold shift to the right in the dose-effect curve for morphine but did not antagonize bremazocine. Although results from the substitutions experiments suggested that a component of the BW373U86 discriminative stimulus was mediated through mu opioid receptors, the fact that naltrindole was 1000-fold more potent and naltrexone was 30-fold less potent in antagonizing BW373U86 than morphine indicated that the discriminative effects of BW373U86 were also mediated through delta opioid receptors.

Amino Acid Sequence↗
PubMed1993

A novel delta opioid agonist, BW373U86, in squirrel monkeys responding under a schedule of shock titration.

The squirrel monkey titration procedure was used to assess the antinociceptive effects of the novel delta opioid agonist (+/-)-4-(a-R*)-a(2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxyb enzyl)- N,N-diethylbenzamide (BW373U86). Under this procedure shock increased every 15 sec from 0.01 to 2.0 mA in 30 steps. Five responses terminated the shock for 15 sec, after which the shock resumed at a lower intensity. The intensity at which the monkeys maintained the shock 50% of the time (median shock level, MSL) was determined. BW373U86 (1.0-30.0 mg/kg i.m.) increased MSL, but these increases were not dose-dependent and lasted only 15 min or less. Doses of BW373U86 that increased MSL often produced tremors and/or convulsions immediately after administration. When 1.0 mg/kg of naltrindole, a delta opioid antagonist, was given in combination with BW373U86, doses of BW373U86 up to 30 mg/kg no longer increased MSL nor did tremors and/or convulsions occur. Doses of BW373U86 (0.01-0.3 mg/kg i.m.) that did not increase MSL when administered alone shifted the dose-effect curve for the mu agonist l-methadone to the left. These shifts were antagonized dose-dependently by naltrindole. In monkeys that were tolerant to morphine, BW373U86 (0.03-0.1 mg/kg i.m.) shifted the morphine dose-effect curve leftward. In addition, BW373U86 altered the effects of the partial opioid agonists, buprenorphine, nalbuphine, butorphanol and levallorphan such that doses of these drugs that did not increase MSL when administered alone, often did so in the presence of BW373U86. Taken together, these findings indicate that BW373U86 has a delta agonist profile in the squirrel monkey; however, its antinociceptive effects in the shock titration procedure may be due to its toxic effects.

Amino Acid Sequence↗
PubMed1993

A nonpeptidic delta opioid receptor agonist, BW373U86, attenuates the development and expression of morphine abstinence precipitated by naloxone in rat.

The effect of (+-)-4-((alpha-R*)-alpha-((2S*,5R*)-4-allyl-2,5-dimethyl-1- piperazinyl)-3-hydroxybenzyl)-N,N-diethylbenzamide (BW373U86), the first potent nonpeptidic, highly selective delta opioid receptor agonist, on morphine dependence was studied in rats. Continuous infusion of BW373U86 by a subcutaneously implanted osmotic minipump did not induce any abnormal behavior. After 6 days of BW373U86 infusion, intraperitoneal injection of a high dose of naloxone or naltrindole did not precipitate morphine-like abstinence syndromes. Furthermore, a single injection of BW373U86 did not induce abstinence syndromes or modulate morphine abstinence precipitated by naloxone in chronic morphine-treated rats. However, naloxone-precipitated abstinence syndromes in morphine-dependent rats were partially suppressed by BW373U86 in a dose-dependent manner when the compound was infused subcutaneously before and throughout morphine treatment. Abstinence signs such as wet-dog shake, forelimb tremor and teeth chattering were either suppressed or the intensity was significantly attenuated in these BW373U86-infused rats. This effect was antagonized by naltrindole. These data show that chronic infusion of BW373U86 does not produce physical dependence and that it attenuates some abstinence behaviors in morphine-dependent rats via delta opioid receptors.

Animals↗
PubMed1993

Convulsive effects of systemic administration of the delta opioid agonist BW373U86 in mice.

A systemically active, nonpeptidic delta receptor-selective agonist, (+-)-4-((alpha-R*)-alpha-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl) -3- hydroxybenzyl)-N,N-diethylbenzamide (BW373U86), produced a brief, nonlethal convulsion in mice. The behavioral pattern of convulsion produced by pentylenetetrazol was similar to that produced by systemic administration of BW373U86. Although several episodes of convulsion occurred with pentylenetetrazol, BWB373U86 produced a single, brief episode. Naltrexone (10.0 and 100 mg/kg) and naltrindole (1.0, 3.2 and 10.0 mg/kg), but not midazolam (0.32 mg/kg), produced dose-dependent rightward shifts in the potency of BW373U86 to induce a convulsion. A dose of 3.2 mg/kg of midazolam completely eliminated convulsions induced by BW373U86. Midazolam (0.32 and 3.2 mg/kg), but not naltrindole (3.2 and 32.0 mg/kg), produced parallel rightward shifts in the pentylenetrazol dose-effect curve. Pretreatment with a single injection of BW373U86 (3.2, 10.0, 32.0 or 100 mg/kg) produced a dose-related reduction in the capacity of BW373U86 to induce a second convulsion. Recovery of sensitivity to BW373U86 did not return to control levels for up to 2 weeks after pretreatment with a single injection of 32.0 mg/kg of BW373U86. Naltrindole (3.2 mg/kg) administered within 1 hr, but not at 2 hr, after a pretreatment dose of 10.0 mg/kg of BW373U86 prevented the refractoriness (tolerance) induced by the single dose of BW373U86. These data suggest that the convulsions as well as the tolerance induced by BW373U86 were initiated through delta opioid receptors.

Amino Acid Sequence↗
PubMed1993

Alternative transcript of the nonselective-type endothelin receptor from rat brain.

A novel cDNA encoding the nonselective type of endothelin (ET) receptor was isolated from a rat brain cDNA library. The cloned cDNA encoded a 442-amino acid protein with seven putative transmembrane domains. Nucleotide sequence analysis showed that the rat brain cDNA differed from the cloned rat lung nonselective ET receptor (ETB) cDNA by three extra nucleotides in its coding regions, which produced an encoded protein with four amino acid substitutions. In addition, both the 5' and 3' noncoding sequences of the rat brain cDNA were divergent from those of rat lung cDNA. Expression of the rat brain cDNA in COS-1 cells demonstrated that the encoded receptor displayed equal affinity toward the three ET isopeptides. However, Southern blot analysis indicated a single-copy gene for the rat ETB receptor. Further genomic cloning and sequence analysis demonstrated that rat brain cDNA encoded the authentic protein sequences of the rat ETB receptor. Moreover, the 5' noncoding sequences in rat brain cDNA that were divergent from those in rat lung cDNA were encoded by a distinct region, an upstream exon, in the rat ETB genome. All the findings suggest that rat brain cDNA represents an alternative transcript of the rat ETB gene. Preliminary Northern blot analysis indicated that the expression of this ETB cDNA sequence might be not only in the brain but also in other tissues, whereas its expression might be somehow tissue-specifically regulated.

Alternative Splicing↗
PubMed1993

Comparison of DNA stemline and cell kinetics between primary breast cancer and its lymph node metastasis.

Quantitative measurement of nuclear DNA in individual cells was performed on imprint smears of surgical specimens from 25 primary breast cancers and their concurrent regional lymph node metastases (LNMs). Twenty-five paired imprints taken from primary tumors and their LNMs were stained with the modified Feulgen method. The DNA content was measured with a scanning microdensitometer at a wavelength of 550 nm. The status of estrogen receptors in the primary tumor was measured with the charcoal-dextran-coated method. The DNA modal values were identical in 22 (88%) of 25 paired imprints. Among 13 estrogen receptor-positive (ER[+]) tumors, the DNA modal values were diploid or near-diploid (< 2.5C) in 8 (62%) primary tumors and 7 LNMs. In 10 estrogen receptor-negative (ER[-]) tumors, only 1 (10%) LNM had a near-diploid DNA modal value; all others were aneuploid. In primary tumors the DNA modal values of ER(+) tumors were significantly different from those of ER(-) tumors. The S-phase fraction value of a primary tumor correlated significantly with that of its LNMs. The percentage of cells with DNA content > 6.0C correlated highly between the primary tumor and its LNM. Of primary tumors, ER(-) tumors had more cells with high DNA content (> 6.0C) than ER(+) tumors did. A great similarity in cumulative DNA histograms between primary tumors and their LNMs was found, irrespective of the status of estrogen receptors. In conclusion, the primary breast cancer and its LNM had similar DNA distribution patterns--e.g., DNA modal value, S-phase fraction value and percentage of cells with high DNA content.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy↗
PubMed1992

The antibiotic azatyrosine suppresses progesterone or [Val12]p21 Ha-ras/insulin-like growth factor I-induced germinal vesicle breakdown and tyrosine phosphorylation of Xenopus mitogen-activated protein kinase in oocytes.

The antibiotic azatyrosine [DL-3-(5-hydroxy-2-pyridyl)alanine] suppressed meiotic maturation in oocytes induced by progesterone or the combination of [Val12]p21Ha-ras microinjection and insulin-like growth factor I. The suppression was dose-dependent in the range of 20-250 microM azatyrosine. In addition, azatyrosine blocked the tyrosine phosphorylation of Xp42, a member of the mitogen-activated protein kinase family, after progesterone or [Val12]p21Ha-ras/insulin-like growth factor I stimulation. Activation of maturation-promoting factor, as shown by a decrease in the tyrosine phosphorylation of the Xenopus homolog of p34cdc2, was also suppressed by azatyrosine. Azatyrosine had no effect in vivo or in vitro on the growth factor-induced autophosphorylation of the oocyte insulin-like growth factor I receptor. Azatyrosine has been shown by others [Shindo-Okada, N., Makabe, O., Nagahara, H. & Nishimura, S. (1989) Mol. Carcinog. 2, 159-167] to inhibit the growth of ras-transformed cells without affecting that of nontransformed cells. In oocytes, the antibiotic exerts an inhibitory action on both a ras-dependent and a ras-independent pathway. Lack of an effect of azatyrosine on germinal vesicle breakdown induced by the microinjection of an extract from mature oocytes, however, suggests that azatryosine is acting upstream of maturation-promoting factor activation.

Amino Acid Sequence↗
PubMed1992

Inhibition of phosphatidylinositol 4-phosphate kinase by heparin. A possible mechanism for the antiproliferative effects of heparin.

Heparin and related glycosaminoglycans are important modulators of vascular smooth muscle cell growth, and may be involved in pathological processes such as atherosclerosis. Since polyphosphoinositide metabolism is a major mechanism for regulating cellular activities, including proliferation, the effects of glycosaminoglycans and polyanionic compounds on the activities of phosphoinositide kinases were characterized. Heparin and heparan sulphate caused dose-dependent inhibitions of rat brain cytosolic phosphatidylinositol 4-phosphate (PIP) kinase activity, with half-maximal inhibitory concentrations of approx. 0.5 and 5 microM respectively. PIP kinase was also inhibited by several dextran sulphates, but was not sensitive to inhibition by keratin sulphate, chondroitin sulphate or hyaluronic acid. Polynucleotides and acidic polypeptides were only weakly inhibitory. Heparin did not alter either the PIP- or the Mg(2+)-dependence of PIP kinase. Addition of heparin to brain membranes suppressed PIP kinase activity without affecting phosphatidylinositol (PI) kinase activity. Heparin interfered with the ability of a GTP analogue to stimulate PIP kinase activity in these membranes, suggesting that it uncouples the kinase from an activating guanine-nucleotide-binding protein. In cultured A-10 vascular smooth muscle cells, heparin caused dose- and time-dependent inhibition of [3H]thymidine incorporation into DNA. Similar treatments with heparin decreased cellular levels of phosphatidylinositol 4,5-bisphosphate (PIP2) without changing PI and PIP levels. Therefore heparin-mediated inhibition of PIP kinase appears to lead to decreases in PIP2 levels which may attenuate cellular proliferation.

Animals↗
PubMed1992

Purification of opioid receptor in the presence of sodium ions.

Opioid receptor was solubilized from rat brain membranes with a mixture of the detergents CHAPS and digitonin in the presence of protease inhibitors and 1 M NaCl. The solubilized receptor bound mu-opioid agonists and antagonists with affinities similar to those of native membrane receptor. The affinity of solubilized receptor for the agonist PL017 was greatly reduced by GTP gamma S, suggesting the receptor is still associated with G-protein. The solubilized material was passed through an opioid antagonist (10cd) affinity column and a wheat germ agglutinin column, set up in series, to obtain a partially purified receptor preparation. This partially purified material bound mu-agonist with low affinity and the binding affinity was no longer affected by GTP gamma S. The partially purified receptor was further purified by repeating the affinity and lectin chromatography with smaller size column. Binding of opioid antagonist [3H]diprenorphine to the partially or purified receptors was dependent upon the presence of sodium ions. The purified receptor showed saturable and stereospecific binding for opioid ligands, was predominantly of the mu-type, and exhibited as a diffuse band with a medium molecular mass of 62 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The average specific binding activity of the purified receptor was 18.8 +/- 2.3 pmol/micrograms protein, a value close to the theoretical estimation.

Animals↗
PubMed1992

Induction of ovulation and oocyte maturation of amphibian (Rana dybowskii) ovarian follicles by protein kinase C activation in vitro.

We previously reported that protein kinase C (PKC) activation induced meiotic maturation (germinal vesicle breakdown, GVBD) of Rana dybowskii follicular oocytes cultured in vitro without hormone treatment. The experiments reported here were carried out to establish whether ovarian follicles ovulated in response to PKC activation during culture. A phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was used for PKC activation. TPA addition (10 microM) to cultured ovarian fragments induced ovulation and maturation of the oocytes similar to that seen following addition of frog pituitary homogenate (FPH, 0.05 pituitary/ml) or progesterone (0.5 microgram/ml). Such changes were not observed when ovarian fragments were treated with inactive phorbol ester. The time course of TPA-induced ovulation was similar to that produced by FPH-stimulated ovulation. Both TPA- and FPH-stimulated ovulation and maturation were blocked by treatment with cycloheximide, forskolin (an adenylate cyclase stimulator), and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7; a PKC inactivator). FPH treatment markedly increased progesterone levels in the medium during ovarian fragment culture whereas TPA treatment failed to elevate progesterone levels. Thus, TPA treatment mimics FPH and progesterone in inducing ovulation and meiotic maturation in cultured amphibian ovarian fragments. The data strongly suggest that PKC plays an important role in regulating ovulation as well as in modulating amphibian oocyte maturation during follicular differentiation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗