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Biomedical subjects

R J Chang

Publications and source records attributed to R J Chang.

At least 19 recordsLinked to original sources
PubMed1992

Purification and characterization of transforming growth factor-beta 2.3 and -beta 1.2 heterodimers from bovine bone.

A unique form of transforming growth factor-beta (TGF-beta), TGF-beta 2.3 heterodimer, has been purified from bovine bone extract. TGF-beta 2.3 migrated as a single 25-kDa band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas under reducing conditions it migrated as a 12.5 kDa band. The TGF-beta 2.3 reacted positively with anti-TGF-beta 2 and anti-TGF-beta 3 antibodies on immunoblots. Equal levels of TGF-beta 2 and TGF-beta 3 sequences were detected by N-terminal sequencing. TGF-beta 2.3 eluted as a single sharp peak by reverse-phase high performance liquid chromatography. However, prior reduction of the protein with dithiothreitol resulted in the protein eluting in two peaks, one containing predominantly TGF-beta 3 and the other containing predominantly TGF-beta 2. TGF-beta 2.3 inhibited proliferation of mink lung epithelial cells and promoted the formation of colonies of normal rat kidney fibroblasts in culture with specific biological activity similar to those of TGF-beta 1 and TGF-beta 2. These results demonstrate that the protein is TGF-beta 2.3 heterodimer, consisting of one polypeptide chain each of TGF-beta 2 and TGF-beta 3 linked by one or more disulfide bonds. In addition, TGF-beta 1.2 heterodimer, previously found only in porcine platelets, has also been purified from bovine bone extract.

Amino Acid Sequence↗
PubMed1992

Long-term administration of gonadotropin-releasing hormone agonist and dexamethasone: assessment of the adrenal role in ovarian dysfunction.

OBJECTIVE: To examine the possible impact of abnormal adrenal steroidogenesis on the ovarian dysfunction seen in polycystic ovarian disease (PCOD). DESIGN: Prospective analysis of blood sampling monthly for 6 months, then three times weekly for 90 days. SETTING: Tertiary institutional outpatient care. PARTICIPANTS: Six anovulatory women with a diagnosis of PCOD. INTERVENTION: Six-month suppression with gonadotropin-releasing hormone agonist (GnRH-a) followed by suppression with dexamethasone (DEX) for 90 days. MAIN OUTCOME MEASURES: Serum levels of testosterone (T), androstenedione (A), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), cortisol, estradiol (E2), progesterone (P), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and bioactive LH. RESULTS: Gonadotropin-releasing hormone agonist administration suppressed greater than 60% of the circulating levels of T and A, suggesting an ovarian origin. Minimal changes of DHEA, DHEAS, and cortisol were seen. With the addition of DEX, there was greater than 90% suppression of the total circulating A, T, DHEA, DHEAS, and cortisol, supporting the adrenal origin of the non-GnRH-a suppressible androgens. Excessive ovarian T and A secretion returned during the 90-day recovery study period in spite of rises of FSH concentrations that changed the ratio of FSH to LH in all subjects. Four of the six women failed to ovulate. In comparison of the women who did and did not ovulate during recovery, no differences in absolute levels or changes in concentrations of steroids or gonadotropins could be detected. CONCLUSIONS: Using sequential and simultaneous administration of GnRH-a and DEX, we were able to delineate the contributions of the ovaries and adrenals to the abnormal steroidogenesis seen in PCOD. Despite prolonged suppression of ovarian and then adrenal steroidogenesis, ovarian dysfunction, evidenced by abnormal androgen production, returned with cessation of agonist administration.

Adrenal Glands↗
PubMed1992

Granulosa cells of polycystic ovaries: are they normal or abnormal?

Understanding whether granulosa cells are normal or abnormal in women with polycystic ovary syndrome (PCO) could have clinical importance. For this purpose, we compared the ability of normal and PCO granulosa cells to synthesize oestrogen and progesterone in vitro in response to follicle stimulating hormone (FSH) and/or insulin-like growth factor-I (IGF-I). The normal granulosa cells were from a 7 mm dominant follicle from a women with regular menstrual cycles. The PCO granulosa cells were from 5-7 mm follicles of three patients who had classical PCO. Several interesting points emerge from the comparison: in each PCO patient there was a high level of bioactive FSH in the follicular microenvironment (greater than or equal to 5 mIU/ml; greater than or equal to 250 ng/ml). This is paradoxical because the concentration of steroids in follicular fluid suggests that PCO follicles are highly atretic and therefore should not contain detectable FSH activity. The capacity to secrete progesterone when challenged with a maximally effective dose of FSH and/or IGF-I, was markedly reduced (8- to 10-fold) in PCO compared to normal granulosa cells. This is in sharp contrast to the oestradiol responses which were much the same for PCO and normal granulosa cells. Also, the time course and dose-response effects of FSH showed some major differences between normal and PCO cells, that is, PCO cells lost their capacity to produce oestradiol when treated continuously with a maximally effective dose of FSH. They were also significantly more sensitive to FSH and failed to become more sensitive to IGF-I when treated with FSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1992

Oxidation of a specific methionine in thrombomodulin by activated neutrophil products blocks cofactor activity. A potential rapid mechanism for modulation of coagulation.

Endothelial thrombomodulin (TM) plays a critical role in hemostasis as a cofactor for thrombin-dependent formation of activated protein C, a potent anticoagulant. Chloramine T, H2O2, or hypochlorous acid generated from H2O2 by myeloperoxidase rapidly destroy 75-90% of TM cofactor activity. Activated PMN, the primary in vivo source of biological oxidants, also rapidly inactivate TM. Oxidation of TM by PMN is inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase. Both Met291 and Met388 in the six epidermal growth factor-like repeat domain are oxidized; however, only substitutions of Met388 lead to TM analogues that resist oxidative inactivation. We suggest that in inflamed tissues activated PMN may inactivate TM and demonstrate further evidence of the interaction between the inflammatory process and induction of thrombotic potential.

Adult↗
PubMed1991

Transforming growth factor-beta 2 enhances the osteoinductive activity of a bovine bone-derived fraction containing bone morphogenetic protein-2 and 3.

We previously identified a novel glycoprotein in an osteoinductive fraction from bovine bone (Bentz et al.: Amino acid sequence of bovine osteoinductive factor. J. Biol. Chem. 265: 5024-5029, 1990). We now find that this fraction also contained small amounts of bone morphogenetic protein-2 and 3 (BMP-2 + 3) previously identified by others (see Wozney, J.M.: Bone morphogenetic proteins. Prog. Growth Factor Res. 1: 267-280, 1990). Separation of BMP-2 + 3 from the glycoprotein was achieved with a modified reversed phase-high pressure liquid chromatographic procedure. When assayed in the rat subcutis using a collagen-ceramic carrier, the osteoinductive activity was found in the subfraction containing BMP-2 + 3. This activity was potentiated and the ratio of cartilage to bone was increased by transforming growth factor-beta 2. The glycoprotein, originally called osteoinductive factor, has been renamed osteoglycin. In its precursor form, osteoglycin is a member of the leucine-rich family of proteins showing the characteristic 24-residue internal homology. Its biological function is unknown.

Amino Acid Sequence↗
PubMed1991

6-Pentadecylsalicylic acid: an antithrombin component isolated from the stem of Rhus semialata var. roxburghii.

Bioassay-directed fractionation of the n-hexane extract of the stem of Rhus semialata Murr. var. roxburghii DC (Anacardiaceae) has led to the isolation of 6-pentadecylsalicylic acid. It showed antithrombin activity at 50 micrograms/ml in the amidolytic method. It also prolonged the clotting time in a dose-dependent manner in the clotting assay of thrombin-fibrinogen interaction.

Amino Acid Sequence↗
PubMed1990

Amino acid sequence of bovine osteoinductive factor.

The complete amino acid sequence of bovine osteoinductive factor (OIF) was determined by automated Edman degradation of S-pyridylethylated bovine OIF and selected fragments. Cleavage with endoproteinase Lys-C, endoproteinase Glu-C, or endoproteinase Asp-N established all fragments in an unambiguous sequence. Bovine OIF contains 105 residues with a calculated molecular weight of 12,055. It is a single chain polypeptide containing two intramolecularly linked cysteines at residues 62 and 95. Two asparagine-linked glycosylation sites at positions 52 and 65 were found by comparing sequence data and peptide profiles of native and deglycosylated OIF fragments. The amino acid sequence of OIF has no homology to other reported proteins.

Amino Acid Sequence↗
PubMed1990

The effects of insulin and insulin-like growth factors-I and -II on estradiol production by granulosa cells of polycystic ovaries.

The objective of this work was to examine the effects of insulin-like growth factors (IGFs) on estradiol (E2) production by granulosa cells obtained from ovaries of patients with polycystic ovary disease (PCO). Granulosa cells, isolated from ovaries of three PCO patients, were cultured in serum-free medium containing either androstenedione alone (10(-7) M) or androstenedione plus graded doses of FSH, IGF-I, IGF-II, and/or insulin. At the end of the culture period (2, 4, or 6 days) E2 levels in the medium were measured by RIA. The results from each patient were similar, and therefore, the data were pooled. In the 6-day time-course experiments, the control (untreated) cells produced relatively high levels of E2 at 2 days; however, none was detected thereafter. Treatment with FSH (30 ng/mL) stimulated E2 production 4-fold at 2 days, but the stimulatory effects of FSH were not sustained during culture. IGF-I at 30 ng/mL mimicked the effects of FSH. Concomitant treatment with FSH and IGF-I caused synergistic increases in E2 production (3-, 13-, and 33-fold at 2, 4, and 6 days, respectively). Dose-response studies revealed that FSH and IGF-I stimulated E2 production in a dose-dependent fashion (ED50 of FSH and IGF-I, were 1.1 +/- 0.3 and 7.6 +/- 7.2 ng/mL, respectively). In the presence of a maximally effective dose of FSH (30 ng/mL), the cells appeared to become more responsive to IGF-I (ED50 of IGF-I plus FSH, 1.09 +/- 0.29 ng/mL); however, this effect was not significant (P = 0.086). In the presence of a maximally effective dose of IGF-I (30 ng/mL), the stimulatory effect of FSH on E2 production was dramatically amplified, but the IGF-I did not significantly (P = 0.85) change the potency of FSH (ED50 of FSH plus IGF-I, 1.07 +/- 2.3 ng/mL). Treatment with IGF-II over the concentration range of 0.1-100 ng/mL had no effect on either control or FSH-stimulated E2 production. Treatment with insulin, either alone or together with FSH, increased the levels of E2, but the insulin effects were seen only at the highest doses tested (0.3-10 micrograms/mL). The results in these in vitro experiments with PCO granulosa cells indicate that 1) physiological concentrations of IGF-I are as effective as FSH in stimulating E2 production; 2) IGF-I and FSH act synergistically to control the level of E2 production; and 3) this synergy was not observed with insulin or IGF-II.

Adult↗
PubMed1989

Subunit composition of the purified dihydropyridine binding protein from skeletal muscle.

The dihydropyridine (DHP) receptor from rabbit skeletal muscle has been characterized by affinity labeling and purification. Two procedures were used for purification: one that was a procedure modified from that of Curtis and Catterall (1984) and one that employed an anti alpha 1 monoclonal antibody (Mab) affinity column. In addition, both digitonin and CHAPS solubilizations were utilized with each purification technique. The major findings are as follows: (1) In contrast to the behavior in digitonin, neither the 52K (beta) nor the 140K (alpha 2) polypeptide quantitatively copurifies with the 170K (alpha 1) polypeptide when the purification is carried out in CHAPS. This has been shown by use of both wheat germ and monoclonal antibody columns. The digitonin-extracted receptor complex bound to the Mab affinity column loses alpha 2 and beta when the digitonin is replaced by CHAPS, and when the complex is bound to a WGA column, a CHAPS wash causes dissociation of alpha 1, beta, and gamma from alpha 2. Loss of binding of dihydropyridines occurs with the CHAPS wash but can be partially restored by the addition of the CHAPS wash to the material eluted from the column with N-acetylglucosamine. (2) Although both detergents solubilized greater than 80% of the polypeptides associated with the DHP binding site, the ability of these proteins to bind dihydropyridines is reduced more by CHAPS treatment than by digitonin treatment, raising the possibility that subunit interactions contribute to high-affinity binding. Alternatively, CHAPS may remove tightly bound lipids necessary for binding or cause irreversible denaturation of the binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1989

Effects of gonadotropin-releasing hormone (GnRH) agonist on pituitary and ovarian responses to pulsatile GnRH therapy in polycystic ovarian disease.

Nine clomiphene citrate-resistant polycystic ovarian disease (PCOD) patients received intravenous gonadotropin-releasing hormone (GnRH) pulses before and immediately after 1 month of GnRH agonist (GnRH-a) therapy. Circulating gonadotropin and ovarian steroid levels, as well as follicular development, were measured throughout therapy. Results were compared with those obtained from five hypogonadotropic patients treated with GnRH pulses only who ovulated during six of seven treatment cycles. Only two PCOD patients ovulated normally with GnRH pulses before GnRH-a therapy. Aberrant gonadotropin and ovarian steroid secretory patterns were noted in the others. After GnRH-a, gonadotropin and ovarian steroid hormone levels were similar to those of the hypogonadotropic patients. Subsequent secretory responses to GnRH pulses were partially normalized. However, only two additional PCOD patients ovulated.

Amenorrhea↗
PubMed1989

CV 205-502 treatment of hyperprolactinemia.

CV 205-502 is a nonergot oral dopamine agonist with specific D2 activity, which has a prolonged suppressive effect on serum PRL and may have fewer side-effects than other dopamine agonists. We treated 26 hyperprolactinemic women with this compound given as a single bedtime (hs) dose for up to 12 weeks. All had gonadal dysfunction, either amenorrhea or oligomenorrhea, and 15 had galactorrhea. The initial and subsequent doses were administered in a randomized fashion; the initial dose ranged from 0.01-0.05 mg, and the dose at 12 weeks ranged from 0.03-0.09 mg. The women were evaluated every 2 weeks, and the dose was increased by 0.02 mg every 4 weeks if the serum PRL level was greater than 20 micrograms/L. Of the 26 women initially enrolled, 24 completed 12 weeks of therapy, and 2 discontinued therapy because of side-effects. Thirteen women (54%) had return of menses, and 12 (80%) had either a decrease in or disappearance of galactorrhea. Serum PRL concentrations decreased to a variable degree in all patients; 13 (54%) achieved a normal serum PRL level (less than or equal to 20 micrograms/L). The mean (+/- SE) pretreatment serum PRL concentration was 129 +/- 34, and it was 29.9 +/- 5.9 micrograms/L after 12 weeks of treatment (P = 0.005). The mean (+/- SE) percent reduction in serum PRL was 66.5 +/- 5.0% (median, 78.0%). A dose response was not demonstrated (r = -0.08; P = 0.70) among the 6 dose groups during the last 4 weeks of therapy. In 5 women, serum PRL levels, measured frequently for 24 h after treatment remained low. Side-effects after the initiation of therapy included nausea, headache, and morning fatigue in 10 women. These symptoms caused 2 women to discontinue therapy; they subsided in the other women. An optimal dose was not determined and will probably need to be determined by titration in each patient. CV 205-502, given once daily, appears to be a safe and effective alternative to other dopamine agonists in the treatment of hyperprolactinemia.

Amenorrhea↗
PubMed1989

Recovery of hormone secretion after chronic gonadotropin-releasing hormone agonist administration in women with polycystic ovarian disease.

Persistent suppression of gonadotropin and ovarian steroid production can be achieved in women with polycystic ovarian disease (PCO) by daily administration of a long-acting GnRH agonist (GnRHa). This study was designed to determine the patterns of recovery of clinical responses and hormonal secretion after chronic GnRHa administration in women with PCO. Six women with PCO were treated with daily sc injections of [D-His6(imBzl),Pro9-NEt]GnRHa (100 micrograms) for 6 months. Blood samples were obtained at the time of and three times weakly for 90 days after discontinuation of agonist therapy. In five women who did not ovulate, the suppressed serum FSH levels rose to pretreatment values within 10 days. In contrast, a gradual and progressive increase in serum LH (as measured by bioassay and immunoassay) was apparent by day 18. The LH increase coincided with progressive increases in serum estrone (E1), androstenedione, and testosterone. Serum estradiol (E2) began to rise on day 28. All hormones returned to their pretreatment baseline values within the 90-day recovery interval, with the exception of E2. Trend analysis of the slopes of recovery revealed that the incremental secretion patterns of E1, E2, androstenedione, and testosterone differed significantly from that of FSH, but not from those of bioactive or immunoactive LH. Serum progesterone, dehydroepiandrosterone sulfate, and cortisol did not change after withdrawal of GnRHa. One woman ovulated spontaneously on day 52 before which her hormone secretion patterns were indistinguishable from those of the other women. In summary, 1) during recovery after discontinuation of chronic GnRH agonist therapy the patterns of FSH and LH release suggested resumption of endogenous GnRH action on the pituitary with greater release of FSH than LH, a pattern that would be expected in the absence of ovarian steroid influence; 2) the lack of early estrogen production despite the increase in serum FSH concentrations suggests inadequate FSH secretion, abnormal ovarian responsiveness to FSH, or impaired FSH bioactivity; 3) androgen secretion was provoked by the increase in LH secretion; 4) per unit LH measured by bioassay, greater ovarian androgen secretion was stimulated in PCO than ovulatory women; and 5) the likelihood of spontaneous ovulation during recovery was minimal.

Adult↗
PubMed1989

A pharmacodynamic comparison of human urinary follicle-stimulating hormone and human menopausal gonadotropin in normal women and polycystic ovary syndrome.

We performed a pharmacodynamic comparison of human urinary follicle-stimulating hormone (hFSH) and human menopausal gonadotropin (hMG) to characterize differences in the bioavailability of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as to compare estrogen responses in normal women and those with polycystic ovary syndrome (PCOS). Ten women with PCOS and ten normal ovulatory controls were randomized to receive a single dose (2 ampules) of either hFSH or hMG. Serum LH decreased significantly following hFSH with responses occurring earlier in controls (24.5 +/- 10.9% after 30 minutes) than in PCOS patients (27.3 +/- 7.5% after 18 hours). After hMG, LH increased only in controls (33.8 +/- 16.3%). An FSH increment following hFSH was observed in both PCOS patients (54.7 +/- 24.8%) and controls (74.6 +/- 36.8%), with peak responses at 6 and 4 hours, respectively. However, after hMG, FSH increased only in controls. The LH/FSH ratio after hFSH decreased, with the nadir at 18 hours (1.438 +/- 0.183) being similar to baseline LH/FSH ratios of controls (1.433 +/- 0.341). Serum estradiol (E2) increased following hMG, with peak responses after 18 hours, in both PCOS patients (75.4 +/- 28.6%) and controls (88.5 +/- 32.5%). The peak E2 response to hFSH was observed to be earlier in PCOS patients (147 +/- 34%), occurring after 12 hours, compared with controls (58 +/- 29% after 18 hours).

Adult↗
PubMed1988

Regulation of the number of alpha-bungarotoxin binding sites in cultured chick myotubes by a 1,4 dihydropyridine calcium channel antagonist.

Calcium has been suggested as the second messenger link between skeletal muscle activity and AChR gene expression and synthesis. We have compared the concentrations of the Ca2+ channel antagonists D600 and nisoldipine needed both to block Ca2+ uptake into cultured myotubes and to increase AChR expression. The good correspondence between these two measurements and the use of the highly specific Ca2+ channel antagonist nisoldipine strengthens the hypothesis that AChR expression is regulated by levels of intracellular Ca2+.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1988

Identification of a approximately 170K subunit of the cardiac calcium channel using a monoclonal antibody to the skeletal muscle dihydropyridine receptor.

Our newly isolated monoclonal antibody (#78) specifically interacts with the 170Kd 1,4 dihydropyridine binding component of the skeletal muscle calcium channel. Dihydropyridine receptor (DHPR) from rabbit skeletal muscle and canine cardiac membranes were purified by monoclonal antibody #78 affinity chromatography. We show that DHPR from canine cardiac membranes like DHPR from rabbit skeletal membranes contain a approximately 170Kd polypeptide to which antibody #78 immunoblots under both reducing and non-reducing conditions.

Animals↗
PubMed1988

An assay using embryo aggregation chimeras for the detection of nonlethal changes in X-irradiated mouse preimplantation embryos.

We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos.

Animals↗
PubMed1987

Reproductive effects of chlorpromazine exposure to female mice: cell proliferation disadvantage revealed by the Chimera Embryo Assay.

Administration of chlorpromazine-HCl at 5 to 15 mg/kg bodyweight to pregnant CD-1 mice at 24 h after human chorionic gonadotropin (hCG) (20-23 h after mating) inhibited blastocyst formation and reduced the cell number of embryos recovered at 95 h after hCG. When embryos are recovered at the two- to four-cell stage (48-50 h after hCG) and cultured for an additional 47 h (to 95 h after hCG) or 72 h (to 120 h after hCG), blastocyst formation and embryo cell number were similarly reduced. When the dose range was reduced to 0.5 to 2 mg/kg bodyweight, no significant effect of the drug was observed on blastocyst formation or on embryo cell number. However, when aggregation chimeras were formed between embryos recovered from drug-exposed females and from untreated females, a decrease in cell proliferation rate of the embryo from the drug-exposed female was observed at a dose of 2 mg/kg bodyweight. This result indicates that exposing pregnant mice to chlorpromazine-HCl at doses as low as 2 mg/kg bodyweight can induce a potential for decreased cleavage rate in their pre-implantation embryos that can be revealed by challenging those embryos by direct contact with embryos from nonexposed females. Finally, when four-cell stage embryos recovered from untreated females cultured in the presence of chlorpromazine (0.1-25 mM), blastocyst formation and embryo cell number were significantly reduced in a dose-dependent manner. This last result suggests that in vivo the drug may act directly on the embryo from the pronuclear stage to the early morula stage of development.

Animals↗