PubMed Health⌕ Search

Biomedical subjects

D T MacLaughlin

Publications and source records attributed to D T MacLaughlin.

At least 55 records · Page 3Linked to original sources
PubMed1990

An immunoassay to detect human müllerian inhibiting substance in males and females during normal development.

An enzyme-linked immunosorbent assay has been developed to measure human Müllerian inhibiting substance (MIS) in biological fluids. The enzyme-linked immunosorbent assay is specific for MIS, with a sensitivity in human serum to 0.5 ng/ml and does not recognize transforming growth factor-beta 1 or -beta 2, LH, or FSH. It similarly fails to recognize other proteins secreted from the cell type into which the MIS gene was cloned. MIS was detected in the serum of normal newborns, infants, children, and adults. In males the serum level of MIS is 10-70 ng/mL at birth. The level increases slightly after birth, and then decreases to a basal level of 2-5 ng/mL after the first 10 yr of life. Newborn male urine contains minimal amounts of MIS (0.5 ng/mL). In females MIS is barely detectable in serum at birth, but rises to the basal level equal to that seen in males after 10 yr of age. Similar basal levels of MIS were found in adult ovarian follicular fluid. MIS levels were high in the serum of a female patient with a sex cord tumor (3200 ng/mL), but fell to 100 ng/mL after multiple excisional operations. In addition, a serum MIS level of 20 ng/mL was detected in a patient with an ovarian granulosa cell tumor. A sensitive assay for MIS could be useful in the diagnosis of patients with congenital abnormalities of sexual development and patients with Sertoli cell and/or other MIS-producing neoplasms. Other applications may also be recognized as the biology of MIS in both males and females is further elucidated.

Adolescent↗
PubMed1990

Fibroid and myometrial steroid receptors in women treated with gonadotropin-releasing hormone agonist leuprolide acetate.

The reduction in uterine and fibroid volume associated with the chronic administration of a gonadotropin-releasing hormone agonist (GnRH-a) is thought to be secondary to the analogue induced hypoestrogenic state. Our hypothesis was that the concentration of bioactive estrogen receptors (ER) and progesterone receptors (PR) may be important in the regulation of fibroid growth. The purpose of this study was to determine ER and PR content in fibroids and myometria from women pretreated with GnRH-a compared with controls. Tissue was obtained from 20 premenopausal women with uterine fibroids who were randomized to receive either leuprolide acetate depot, 3.75 mg intramuscularly every 28 days for four injections (n = 10) or placebo (n = 10) before myomectomy. The mean fibroid ER and PR content was significantly greater than the mean myometrial ER and PR content. The mean fibroid ER content for GnRH-a-treated patients was significantly greater than in placebo-treated patients (143.3 +/- 22.8 versus 36.1 +/- 14.3 fmol/mg). The mean fibroid PR and the mean myometrial ER and PR content were not significantly different between treatment groups. Clinically, the significant increase in fibroid ER may be an explanation for the rapid regrowth of fibroids observed after the cessation of GnRH-a therapy.

Antineoplastic Agents↗
PubMed1989

In vitro protein production by the human endometrium.

Protein secretion by the human endometrium was studied in vitro in medium after incubation of tissue minces (n = 10) or glands isolated by collagenase digestion (n = 4) from tissues obtained at the time of curettage from normal women. Samples were incubated in a serum-free medium for 24 h at 38 degrees C in the presence of radiolabeled methionine. Dialyzed medium from each sample was subjected to two-dimensional gel separation, and detected by protein staining. Although 5 of the 27 proteins were considered to be present in the labeling experiments by only one of the three observers, there was agreement about the presence of the 22 others. In addition, the observers categorized the proteins into three groups for purposes of analysis: a) those associated with the follicular phase of the cycle; b) those associated with the luteal phase; and c) those not cycle-related. One protein triplet, labeled #27, showed a significant relation to the luteal phase (p less than 0.01). A complete lack of similarity between the pattern of labeled proteins obtained from the medium and labeled proteins obtained from lysates of cells incubated in the same experiments makes it unlikely that cellular lysis, as opposed to secretion, contributed to the pattern of proteins studied in these experiments.

Culture Techniques↗
PubMed1989

Recombinant human müllerian inhibiting substance inhibits epidermal growth factor receptor tyrosine kinase.

Autophosphorylation of the epidermal growth factor (EGF) receptor in A-431 cells and plasma membrane fractions was inhibited by partially purified recombinant human Müllerian Inhibiting Substance (MIS). Immunoprecipitation of the EFG receptor using anti-EGF receptor or anti-phosphotyrosine antibodies, and phosphoamino acid analysis of this receptor, demonstrated that MIS specifically inhibited EGF-induced tyrosine phosphorylation. Inhibition of EGF receptor autophosphorylation by MIS in membrane preparations was not affected by increasing concentrations of EGF, manganese or [gamma-(32)P] ATP. Thus, it is unlikely that MIS competes for EGF binding sites or sequesters substrate. Immunoabsorption of MIS with anti-human MIS antibody blocked the MIS inhibition of EGF receptor autophosphorylation, indicating that the inhibition was due to MIS. Our data suggest that MIS regulates the activity of the EGF receptor tyrosine kinase in A-431 cells.

Amino Acids↗
PubMed1987

Analysis of proteins secreted by the human endometrium in vivo and in vitro.

Human uterine luminal fluids contain over two dozen proteins distinct from those of serum as detected by two dimensional gel electrophoresis and silver-type protein staining. Eighty-one percent of these uterine fluid proteins can be detected in vitro by radiolabeled methionine incorporation studies and the vast majority of these products are epithelial in origin. The major recognizable menstrual cycle phase-dependent change in the protein pattern in these gels was the appearance of a protein group (number 27) of approximately 25,000 mw and pI of 5.8 - 6.3. This group of proteins was found in nearly all mid- and all late secretory phase fluids or culture media and in none obtained earlier in the cycle. As yet, however, it has not been possible to induce these proteins in proliferative specimens in vitro by the addition of estrogens and/or progestins, though studies along these lines are continuing. Although we cannot be certain, it appears as though protein group number 27 is distinct from, but similar in several respects to, other proteins of human endometrium reported in the literature.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Müllerian inhibiting substance blocks autophosphorylation of the EGF receptor by inhibiting tyrosine kinase.

The fetal regressor Müllerian inhibiting substance (MIS), in concentrations as low as picomolar, inhibited the growth of A-431 cells and the autophosphorylation of its epidermal growth factor (EGF) receptor. The inhibition of membrane phosphorylation was due neither to the reduction of the total number of EGF receptor binding sites, nor to stimulation of intrinsic phosphates, but rather to inhibition of tyrosine kinase activity. MIS control of EGF receptor autophosphorylation by tyrosine kinase may be one mechanism by which Müllerian duct regression in the embryo and the inhibition of A-431 proliferation is initiated. In addition, MIS as an inhibitor of phosphorylation may furnish a tool to probe the role of membrane phosphorylation in growth control.

Animals↗
PubMed1986

Two-dimensional gel electrophoresis of endometrial protein in human uterine fluids: qualitative and quantitative analysis.

By combining two-dimensional gel electrophoresis, protein staining and a sensitive computer-assisted gel scanning system, it was possible to examine human uterine fluid (n = 56) qualitatively and quantitatively for the presence of endometrial proteins. The protein concentration of uterine fluids ranged from 0.1 to 12.0 mg/ml with early secretory phase samples (n = 15) having significantly less protein (0.72 +/- 0.2 SEM mg/ml p less than or equal to 0.05) than the proliferative phase (n = 57) samples (1.58 +/- .29 SEM mg/ml). Whole blood contamination of uterine fluid, as measured by hemoglobin content, averaged 6.2 +/- 0.88% throughout the menstrual cycle. Human uterine fluids collected throughout the menstrual cycle were found to contain serum and up to 24 other proteins in addition to those previously described (MacLaughlin and Richardson, 1983). These proteins represent approximately 1% of the total protein in the gels and exhibit isoelectric points from 4.5 to 7.0 and molecular weights in the 26,000 to 60,000 range. These proteins are absent from human serum, which exhibits an identical pattern whether obtained in the proliferative or secretory phase of the menstrual cycle. These secreted endometrial proteins now become the standard against which to compare proteins identified in vitro using organ, gland and cell culture techniques and to characterize proteins that are regulated by steroid hormones in vivo.

Body Fluids↗
PubMed1985

Steroid modulation of Mullerian duct regression in the chick embryo.

The recent demonstrations that Mullerian inhibiting substance (MIS) is present in embryonic chick ovaries (P. K. Hutson, H. Ikawa, and P. K. Donahoe (1981). J. Pediatr. Surg. 16, 822-827), and that exogenous diethylstilbestrol does not significantly inhibit MIS secretion from feminized testes (Hutson et al. (1982) J. Pediatr. Surg. 17, 953-959), suggest that ovarian estrogens protect the female left Mullerian duct from MIS-induced regression. The possibility exists, however, that ovarian MIS may be inactive. This study was designed to see if interference with estrogen action in ovo would allow MIS to cause regression of the female left Mullerian duct. The "antiestrogens," tamoxifen and LY117018, had little effect on the female Mullerian ducts unless given in high doses or with added testosterone (greater than 0.1 mg). Two compounds known to inhibit estrogen synthesis, aminoglutethimide and 4-hydroxyandrostenedione, had no effect on their own, even in high doses (less than 1.0 mg/egg). However, when administered together (0.5 mg each) there was significant disappearance of the lower ends of both Mullerian ducts. Norethindrone, which has been described recently as an aromatase inhibitor (Y. Osawa, C. Yarborough, and V. Osawa (1982). Science (Washington, D. C.) 215, 1249-1251) caused partial regression of the upper end of the left Mullerian duct as well as complete loss of the lower ends of both ducts in the female. These results suggest that the steroid environment is a critical factor in the response of the Mullerian ducts to MIS, and that estrogen blockage may allow endogenous MIS from the ovary to induce partial regression of the Mullerian ducts in the female chick embryo.

Aminoglutethimide↗
PubMed1985

Estrogen and progestin binding by cytosolic and nuclear fractions of human meningiomas.

Frozen tissue samples were obtained from meningiomas in 42 patients. Both cytosolic and nuclear fractions were tested for estradiol and progestin binding using equilibrium binding assays. The results were correlated with the age of the patient and the histological type and cellular density of the tumor. Cytosolic estradiol binding was noted in 25 (60%) of 42 tumors, with eight (19%) of the 42 having levels over 10 femtomoles (fM)/mg protein. Nuclear estradiol binding was detected in 16 (57%) of 28 tumors, with six (21%) of the 28 having levels over 10 fM/mg protein. Cytosolic progestin binding was noted in 16 (73%) of 22 samples, with levels in nine (41%) of 22 being greater than 10 fM/mg protein. There was no correlation between the level of cytoplasmic progestin binding and either the level of cytoplasmic estradiol binding or the level of nuclear estradiol binding. In several specimens, level of cytoplasmic progestin binding in excess of 100 fM/mg protein were found in tissues demonstrating little or no estradiol binding by either the nucleus or the cytosol. This discrepancy differs from the situation found in other hormonally responsive tissues such as breast or uterus, and suggests either a possible derangement of the normal cellular hormonal control mechanism or that the measured hormone binder is a molecule other than a classical hormone receptor.

Adult↗
PubMed1985

Modulation of meningioma cell growth by sex steroid hormones in vitro.

Meningiomas were removed from four patients and estradiol binding was measured in the tumor tissue. Cell cultures were established and an in vitro system was developed to test the biological activity of physiologically relevant concentrations (10(-7) M and 10(-9) M) of estradiol-17 beta, progesterone, and the anti-estrogen, tamoxifen, on the growth of meningioma cells in early culture (passages 3 to 5). Assays of the frozen surgical specimens demonstrated cytosolic estradiol binding, with levels of 0.3 to 26.7 femtomoles (fM)/mg protein, in all four tumors. Nuclear estradiol binding was detected in three tumors, with levels of 16.8 to 39.5 fM/mg protein. In cell culture, estradiol at either 10(-7) M or 10(-9) M consistently stimulated cell growth in all four cultures. When tested alone, progesterone stimulated the growth of all four tumors and tamoxifen stimulated the growth of three of the four tumors, but the levels of stimulation produced by either of these compounds were less pronounced than the level produced by estradiol. When tested in combination with estradiol, progesterone significantly inhibited the growth stimulation produced by estradiol in all four meningioma cultures and tamoxifen significantly inhibited estradiol-induced growth stimulation in three of four cultures. It is not known if these effects are mediated by a hormone receptor or by a hormone binder different from a true receptor, or if they are caused by alterations in cellular metabolism that are independent of specific hormone binding. However, the authors conclude that this in vitro technique can be used to further study the biological activity of hormones on human meningiomas in order to answer these questions.

Aged↗
PubMed1984

Variations in the levels of secretory component in human uterine fluid during the menstrual cycle.

To determine whether variations in the level of secretory component (SC) occur during the menstrual cycle, we compared the amounts of SC in human uterine secretions during the proliferative, secretory, and menstrual phases. Uterine washes were obtained from women (ages: 18-49) with histologically normal endometria by lavage with Gravlee jet wash device using isotonic glycine. SC was measured by a radioimmunoassay which recognizes primarily free SC. Levels of uterine luminal SC, when expressed as the percentage of total wash protein, were highest during the secretory phase, significantly reduced during the proliferative phase and lowest during menstruation. The mean protein concentration in uterine fluids did not vary significantly during the menstrual cycle. Total amounts of SC were also greatest during the secretory phase, averaging two times higher than SC levels in proliferative samples. These results demonstrate that fluctuations do occur in the SC content of human uterine secretions during the menstrual cycle and suggest that hormones play an important role in the secretory immune system of the human uterus.

Adenocarcinoma↗
PubMed1984

KLE: a cell line with defective estrogen receptor derived from undifferentiated endometrial cancer.

KLE is a cell line derived from a poorly differentiated endometrial carcinoma that is aneuploid with chromosome numbers ranging from 51 to 66 and 6-8 marker chromosomes demonstrated by G banding. Tumors harvested from five of five nude mice bearing an inoculum for more than a month resemble the original specimen, and electron microscopy shows microvilli, many junctional processes, glycogenation, and a prominent nucleolonema. The cell cytosol contains a specific binder for estradiol, but there is no estrogen receptor in the nucleus and in a study reported elsewhere (Raam et al., Breast Cancer Res. Treat. 2, 277 (1982) ) translocation to the nucleus fails to occur. The enzyme phenotype of this cell is human, non-HeLa.

Adenocarcinoma↗
PubMed1983

Analysis of human uterine luminal fluid proteins following radiolabeling by reductive methylation: comparison of proliferative and secretory phase samples.

The proteins contained in human uterine luminal washings of proliferative and secretory phase endometria were depleted of albumin by affinity chromatography and labeled with either 3H or 14C by reductive methylation. Samples of human serum from proliferative and secretory phases of the normal menstrual cycle were similarly treated and served as control material. Isotope mixing experiments were performed in which 3H-labeled fluid proteins were combined with 14C-labeled sera or fluid proteins and the mixture analyzed by two-dimensional gel electrophoresis. The labeled proteins were then detected by fluorography and autoradiography of the same gel. Control experiments mixing 3H- and 14C-labeled sera revealed no difference between proliferative and secretory phase samples and no isotope effect of in vitro labeling. Radiolabeled uterine luminal wash proteins, however, did show significant differences when compared to serum. Furthermore, significant differences between proliferative and secretory phase uterine fluid proteins were observed. A cluster of proteins in the pI range of 5.9 to 6.4 pH and in the molecular weight range of 60,000 -- 67,000 daltons appeared to originate from the endometrium. Among these uterine proteins, several seemed to be found only in the secretory phase of the normal menstrual cycle. The data provided evidence to support the hypothesis that the human endometrium secretes proteins that are dependent on the hormones of the menstrual cycle.

Adult↗