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C R Lyttle

Publications and source records attributed to C R Lyttle.

At least 73 records · Page 4Linked to original sources
PubMed1984

Monoclonal antibody to rat uterine peroxidase and its use in identification of the peroxidase as being of eosinophil origin.

Peroxidase was purified from uteri of estrogen-treated rats by calcium chloride extraction, affinity chromatography on concanavalin A-Sepharose and hydrophobic interaction chromatography on phenyl-Sepharose. An overall purification of greater than 1700-fold was achieved with a final recovery of 27%. Monoclonal antibodies to peroxidase were subsequently prepared by immunization of male C57BL/10J mice with the highly purified peroxidase from rat uterus. Spleen and lymph node cells from the mice were fused with Sp2/0-Ag 14 mouse myeloma cells. The resultant hybrid cells were screened for production of antibody using a solid-phase, double antibody radioimmunoassay. The mature rat spleen, shown previously to be abundant in eosinophils, contains high peroxidase activity. Spleen peroxidase purified by the same procedure as the uterine enzyme cross-reacted with a monoclonal antibody, designated IgG-107B, used in all subsequent studies. Peroxidase extracted from isolated rat eosinophils also cross-reacted with the antibody and yielded identical titers as the spleen and uterine peroxidases. Spleen, uterine and horse eosinophil peroxidase had the same apparent molecular weight, 57000, as determined by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis. Following electrophoretic transfer to nitrocellulose, spleen, uterine and eosinophil peroxidase reacted with monoclonal antibody, using an immunoblotting technique. These results provide biochemical and immunological evidence that the majority of the calcium chloride-extractable peroxidase activity from the uteri of estrogen-treated rats is derived from infiltrating eosinophils.

Animals↗
PubMed1984

Eosinophils as the source of uterine nuclear type II estrogen binding sites.

Rat uterine nuclei have been reported to contain two types of estrogen binding sites (I and II). Type I is the classical high affinity, low capacity translocatable estrogen receptor, while type II is a lower affinity non-translocatable binding site. Correlation of data on hormonal specificity of induction and inhibition, tissue and cellular localization, and ontogenic appearance for type II binding sites and eosinophils suggested the hypothesis that these binding sites are associated with eosinophils. Although type II sites are reported to be highly correlated with uterine growth, premature growth can be elicited in newborn rats by five daily estradiol injections without the appearance of type II sites. Under these conditions, no eosinophils, as measured by peroxidase activity, are found. However, multiple estradiol injections on postnatal days 6-10 or 10-14 increased the levels of both type II sites and eosinophils. Eosinophils, purified from a peritoneal lavage, were found to contain a low affinity binding site with characteristics similar to type II binding sites. Short term estrogen-treated rat uteri contain only type I nuclear receptor and low levels of eosinophils while long term estrogen-treated rat uteri contain both type I and type II nuclear binding sites as well as approximately 6 X 10(5) eosinophils/uterus. The addition of this number of purified eosinophils to short term treated uteri resulted in saturation curves and Scatchard plots identical to those seen in long term treated uteri. These data indicate that the type II nuclear binding site is transported into the uterus with eosinophils following estrogen treatment.

Animals↗
PubMed1984

Steroidal regulation of rat uterine in vitro mRNA translation products.

Total rat uterine mRNA was isolated following 1,2 or 3 days of estradiol, 3 days of progesterone, or 3 days of progesterone and estradiol treatment of immature animals. The mRNA translation products were analyzed by 2-dimensional electrophoresis. The results demonstrate that a number of mRNA products were affected by varying the hormonal conditions. Estradiol consistently caused an increase in mRNA in vitro translation products corresponding to polypeptides with mol. wt of 40,000 Daltons (p40), 59,000 Daltons (p59). A polypeptide with a mol. wt of 37,000 Daltons (p37) consistently decreases in concentration following estradiol administration. Progesterone, alone, caused a decrease in p37, p59, and a slight increase in p40. When progesterone was combined with estradiol we detected translation product levels similar to those seen following 1 day of estradiol treatment. Thus, the change in mRNA populations as a response of the immature rat uterus to estradiol and progesterone can be utilized to characterize the tissue's response to these hormones. This type of study also provides a method for detecting gene products which may possibly be utilized as potential markers to investigate estrogen and progesterone action.

Animals↗
PubMed1984

Serum hormones and their receptors in women with normal and inadequate corpus luteum function.

The purpose of this study was to attempt to characterize women with a luteal phase defect (LPD) by cytosol estrogen and progesterone (P) levels and peroxidase activity in endometrial biopsy samples. These values were correlated with circulating serum estradiol, P, testosterone, and dehydroepiandrosterone sulfate measurements. Similar data were obtained from women during the proliferative phase of the cycle. Midluteal phase P values were significantly greater in women with normal luteal function than in those with LPD; however, some overlap of values existed. Furthermore, because no significant differences between this group and normal women were seen, we were unable to characterize women with LPD by estrogen or P receptor levels or endometrial peroxidase activity.

Corpus Luteum↗
PubMed1982

Cytosol and nuclear estrogen and progesterone receptors in the rabbit endocervix.

This report describes the measurement of estrogen and progesterone receptors in cytosols and nuclear fractions from endocervical tissue components. Unoccupied cytosol estrogen receptor levels as determined by Scatchard analysis of [3H]-estradiol binding data indicated a single class of high affinity binding sites for the epithelial-stromal complex (KD = 0.74 x 10(-9) M). Binding was specific for estrogen (estradiol greater than estriol greater than estrone) and unaffected by desoxycorticosterone, dihydrotestosterone and progesterone. Assays for total estrogen receptor verified that 71.6 +/- 5.3% of this 8S estrogen receptor is in the epithelial-stromal complex while the remaining approximately 28% is localized in the stroma and fibromuscular wall, with the cells of the complex containing the highest receptor concentration. In 5-day pseudopregnant and ovariectomized rabbits compared to estrous rabbits there was a 50% decrease in the cytosol estrogen receptor in the epithelial-stromal complex and a 30% decrease in the concentration of nuclear receptor. Cytosol and nuclear progesterone receptors were measured as an indicator of estrogen action in the rabbit endocervix. Cytosol progesterone receptor concentrations (fmol/mg DNA) in 5-day pseudopregnant and ovariectomized animals were reduced to approximately 35% of the concentration in estrous animals. Nuclear progesterone receptor concentrations decreased 65% in 5-day pseudopregnant and 90% in ovariectomized animals suggesting decreased receptor synthesis. Collectively these data support the concept that the rabbit endocervix may be directly regulated by estrogens.

Animals↗
PubMed1981

Estrogen receptor and peroxidase activity in epithelial ovarian carcinomas.

The endocrine biology of cancers originating from the ovarian epithelium was examined with respect to three sequential indicators for estrogen action: available estrogen receptor in the cytosol, total extractable estrogen receptor from the nucleus, and endogenous tissue peroxidase--a putative postnuclear marker for estrogen-induced growth in uteri of laboratory animals and in some mammary tumor models. Cancers of human ovarian epithelium were distinguished from other ovarian tumors by a higher incidence of detectable (greater than 50 fmol/g tissue wet wt) estrogen receptor in the cytosol (P less than 0.001). Approximately one-half (14/29) of the ovarian adenocarcinoma specimens had greater than 500 fmol available estrogen receptor/g tissue wet weight in their cytosols when assayed by a 2-hour incubation with 17 beta-[2,4,6,7-3H(N)]estradiol followed by treatment with dextran-coated charcoal. With a single exception, ovarian adenomas and nondiseased specimens of premenopausal and postmenopausal ovaries (n = 24) contained less than 500 fmol available estrogen receptor/g tissue wet weight in their cytosols. With respect to extractable estrogen receptor in the nucleus, 11/14 primary and 3/9 metastatic ovarian adenocarcinomas had greater than 50 fmol/g wet weight, as assayed by exchange at 30 degrees C for 5 hours after adsorption of the extracted receptor to hydroxylapatite. Endogenous peroxidase activity, measured in vitro by guaiacol oxidation, occurred in substantially higher amounts in the primary ovarian adenocarcinomas than in benign tumors and control ovaries and could be demonstrated within ovarian adenocarcinoma cells by electron microscopy.

Adenocarcinoma↗
PubMed1979

Steroid hormone regulations of uterine peroxidase activity.

Uterine peroxidase enzyme activity has been studied as a marker for estrogen action in the uterus to help clarify the mechanism of estrogen action and its modulation by antiestrogens and progestins. Estrogen-induced increases in peroxidase were found to closely parallel increases in uterine weight and DNA content in the castrate rat. In the cycling female rat, uterine peroxidase levels were highest during proestrus and estrus and the lower levels of metestrous and diestrous uteri could be raised to estrous levels by administration of estrogen. However, the estrous levels were not further increased by estrogen treatment. The antiestrogen, CI628, while a very weak inducer of uterine peroxidase, is an effective antagonist of the estrogen induction of the enzyme. The prolonged duration of this CI628-effected inhibition corresponds to the prolonged depletion of cytoplasmic estrogen receptor seen with CI628 treatment. Progesterone, R5020 and norethindrone were also found to be effective antagonists of estrogen-induced uterine peroxidase. Medrogestone and clogestrone, less potent progestins in the rat, were also less effective antagonists of peroxidase induction. Since progesterone was found to inhibit peroxidase induction due to both estrone and diethylstilbestrol, as well as estradiol, it is considered unlikely that this antagonism relates to progestin-induced increases in uterine 17 beta-hydroxysteroid dehydrogenase. Rather, it is proposed that progestins, acting through progestin receptor, may have a more direct role, possibly acting at the level of the genome to repress the expression of estrogen-induced products.

Animals↗
PubMed1979

Peroxidase activity and iodide uptake in hormone-responsive and hormone-independent GR mouse mammary tumors.

Transplanted mammary tumors growing in the inbred GR/AFib mouse were assayed for peroxidase activity and ability to concentrate injected 125I. Both tumor peroxidase activity and iodide uptake were about ten times greater in the hormone-resonsive (HR) tumors than in the hormone-independent tumors. However, although peroxidases are known for their ability to participate in the iodination of proteins, over 90% of the radioactive iodine found in the tumors was shown to be free iodide. This finding suggests that these two parameters may be independent of each other, but both are higher in HR tumors.

Adenocarcinoma↗
PubMed1978

Isolation and purification of rat mammary tumor peroxidase.

7,12-Dimethylbenz(a)anthracene-induced rat mammary tumors often contain high levels of the enzyme perioxidase, a putative marker of estrogen dependence. This enzyme can be effectively extracted with 0.5 M CaCl2, giving rise to a soluble peroxidase with a molecular weight of about 50,000 as determined by gel filtration. This is the same size as the estrogen-induced peroxidase of rat uterus but smaller than other mammalian peroxidases. Further purification of the rat mammary tumor peroxidase by concanavalin A-Sepharose chromatography and hydrophobic interaction chromatography on phenyl Sepharose provides a 640-fold purification of the enzyme.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1977

Uterine peroxidase as a marker for estrogen action.

Administration of a single dose of estradiol to immature rats gives rise to the appearance of substantial amounts of peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) enzyme activity in the uterus. This enzyme induction, which is inhibited by administration of actinomycin D and cycloheximide, can be detected at 4 hr after administration of estradiol, reaches a maximum level by 20 hr, and thereafter declines. The amount of uterine peroxidase seen at 20 hr after a single dose increases with dose from 0.1 to 100 microgram of estradiol. Estrone and estriol also show dose-dependent induction of peroxidase, and the quantitative peroxidase responses to these steroids follow their uterotropic capacities. The antiestrogen CI628, capable of low levels of enzyme induction by itself, can inhibit the induction due to estrogen. Solubilization of the uterine enzyme with divalent cations, especially calcium, results in a substantially increased yield of peroxidase. This extraction method provides an enzyme of about 50,000 molecular weight in distinction to the large aggregated form obtained by the usual extraction with sodium chloride.

Animals↗
PubMed1977

Pyruvate dehydrogenase complex from higher plant mitochondria and proplastids.

The pyruvate dehydrogenase complex from pea (Pisum sativum L.) mitochondria was purified 23-fold by high speed centrifugation and glycerol gradient fractionation. The complex had a s(20,w) of 47.5S but this is a minimal value since the complex is unstable. The complex is specific for NAD(+) and pyruvate; NADP(+) and other keto acids give no reaction. Mg(2+), thiamine pyrophosphate, and cysteine are also required for maximal activity. The pH optimum for the complex was between 6.5 and 7.5.Continuous sucrose density gradients were used to separate castor bean (Ricinus communis L.) endosperm proplastids from mitochondria. Pyruvate dehydrogenase complex activity was found to be coincident with the proplastid peak on all of the gradients. Some separation of proplastids and mitochondria could be achieved by differential centrifugation and the ratios of the activities of the pyruvate dehydrogenase complex to succinic dehydrogenase and acetyl-CoA carboxylase to succinic dehydrogenase were consistent with both the pyruvate dehydrogenase complex and acetyl-CoA carboxylase being present in the proplastid. The proplastid fraction has to be treated with a detergent, Triton X-100, before maximal activity of the pyruvate dehydrogenase complex activity is expressed, indicating that it is bound in the organelle. The complex had a sharp pH optimum of 7.5. The complex required added Mg(2+), cysteine, and thiamine pyrophosphate for maximal activity but thiamine pyrophosphate was inhibitory at higher concentrations.

Journal Article↗
PubMed1977

Pyruvate dehydrogenase complex from higher plant mitochondria and proplastids: kinetics.

A steady-state kinetic analysis has been performed on the pyruvate dehydrogenase complex from pea (Pisum sativum L.) mitochondria and castor bean (Ricinus communis L.) proplastids. Substrate interaction kinetics for all substrates gave parallel lines consistent with a multisite ping-pong mechanism. Product inhibition studies showed uncompetitive inhibition between acetyl-CoA and pyruvate and competitive inhibition between NADH and NAD(+), both of which are also consistent with this mechanism. In the mitochondrial complex, acetyl-CoA showed noncompetitive inhibition versus CoA which suggests that the intermediate complex is kinetically important in the lipoamide transacetylase component of this complex. In contrast, the proplastid complex showed competitive inhibition in this interaction. NADH is a noncompetitive inhibitor versus CoA in both complexes indicating that these complexes, like the mammalian complex, may have protein-protein interactions between the second and third enzymes of the complex. Since NADH also shows noncompetitive inhibition versus pyruvate, this interaction may extend to all components of the complex. Acetyl-CoA shows noncompetitive inhibition versus NAD(+) which may also be a result of interaction between the second and third enzymes of the complex. The limiting Michaelis constants for substrates and the inhibitor constants for both complexes were determined.

Journal Article↗