Estrogen-induced synthesis of riboflavin-binding protein in immature chicks kinetics and hormonal specificity

The kinetics of estrogen-induced elevation in the plasma concentration of riboflavin-binding protein, a minor yolk constituent, was investigated in immature male chicks, using a specific and sensitive radioimmunoassay proceudre. Following a single injection of the hormone, the plasma riboflavin-binding protein content was enhanced several-fold at 6 h. reaching peak levels around 48 h and declining thereafter. A two-fold amplication of the response was evident on secondary stimulation with the hormone. A 4-h lag phase prior to onset of induction was noticed during both primary and secondary stimulat ions with the steroid hormone. The magnitude of the response was dependent on the hormonal dose whereas the initial lag phase and the time of peak riboflavin-binding protein accumulation were unaltered within the range of hormonal doses tested. The half-life of riboflavin-binding protein in the circulation was 10 h, as calculated from measurement of the rate of disappearance of exogenously administered ¹²⁵I-labelled protein. Simultaneous administration of progestrone did bot affect the kinetics of riboflavin-binding protein production. On the other hand, the antiestrogens, cis- and trans-clomiphene citrates, given 30 min prior to estrogen and cycloheximide, effectively countered the hormone-induced riboflavin-binding protein elaboration. Both progesterone and the anti-esterogens per se were completely ineffective in substituting for estrogen in the inductive ptrocess.     

Estrogen-induced synthesis of riboflavin-binding protein in immature chicks. Kinetics and hormonal specificity

The kinetics of estrogen-induced elevation in the plasma concentration of riboflavin-binding protein, a minor yolk constituent, was investigated in immature male chicks, using a specific and sensitive radioimmunoassay procedure. Following a single injection of the hormone, the plasma riboflavin-binding protein content was enhanced several-fold at 6 h, reaching peak levels around 48 h and declining thereafter. A two-fold amplification of the response was evident on secondary stimulation with the hormone. A 4-h lag phase prior to onset of induction was noticed during both primary and secondary stimulations with the steroid hormone. The magnitude of the response was dependent on the hormonal dose whereas the initial lag phase and the time of peak riboflavin-binding protein accumulation were unaltered within the range of hormonal doses tested. The half-life of riboflavin-binding protein in the circulation was 10 h, as calculated from measurement of the rate of disappearance of exogenously administered 125I-labelled protein. Simultaneous administration of progesterone did not affect the kinetics of riboflavin-binding protein production. On the other hand, the antiestrogens, cis- and trans-clomiphene citrates, given 30 min prior to estrogen and cycloheximide, effectively counteracted the hormone-induced riboflavin-binding protein elaboration. Both progesterone and the antiestrogens per se were completely ineffective in substituting for estrogen in the inductive process.     

Induction of riboflavin-carrier protein in the immature male rat by estrogen: Kinetic and hormonal specificity

The kinetics of estrogen-induced accumulation of riboflavin-carrier protein in the plasma was investigated in immature male rats using a specific and sensitive homologous radio-immunoassay procedure developed for this purpose. Following a single injection of the steroid hormone, plasma riboflavin-carrier protein levels increased markedly after an initial lag period of approximately 24 h, reaching peak levels around 96 h and declining thereafter. A 1.5 fold amplification of the inductive response was evident on secondary stimulation with the hormone. The magnitude of the response was dependent on hormonal dose, whereas the initial lag phase and the time of peak riboflavin-carrier protein induction were unaltered within the range of the steroid doses (0.1–10 mg/ kg body wt.) tested. Simultaneous administration of progesterone did not affect either the kinetics or the maximum level of the protein induced. The hormonal specificity of this induction was further adduced by the effect of administration of antiestrogens viz., En and Zu chlomiphene citrates, which effectively curtailed hormonal induction of the protein. That the induction involvedde novo-protein synthesis was evident from the complete inhibition obtained upon administration of cycloheximide. Passive immunoneutralization of endogenous riboflavin-carrier protein with antiserum to the homologous protein terminated pregnancy in rats confirming the earlier results with antiserum to chicken riboflavin-carrier protein.     

Hormonal modulation of reproduction-specific thiamin carrier protein in the rat

The hormonal modulation of thiamin carrier protein in the plasma and uterine luminal secretion during the normal reproductive phases of the animal (estrous cycle and pregnancy) as well as during experimental estrogenisation was investigated in the rat using a specific and sensitive homologous radioimmunoassay procedure developed for this purpose. Following a single injection of estrogen to immature male rats, thiamin carrier protein rapidly accumulated in plasma attaining peak concentration at 48 h and declining thereafter. A 1.5-fold amplification of the inductive response was observed on secondary stimulation with the hormone. The magnitude of the response exhibited a clear dependency on the dose of the steroid hormone, whereas the time at which peak levels of thiamin carrier protein production was remained unaltered in the concentration range of the steroid tested. The inductive effect of estrogen was severely curtailed by the antiestrogens,viz., En- and Zu-clomiphene citrates, while progesterone was incapable of either modulating the estrogen-induced response or eliciting an induction by itself. Cycloheximide drastically blocked the response to estrogen. Evidence for the ability of uterus to serve as yet another independent site of thiamin carrier protein synthesis was obtained byin vitro incorporation of radioactive amino acids into immunoprecipitable thiamin carrier protein in the tissue explants of estrogenised female rats. The levels of thiamin carrier protein in uterine luminal fluid measured during estrous cycle, pregnancy and experimental estrogenisation exhibited remarkable similarity to the plasma thiamin carrier protein profiles.     

An ABI3-interactor of conifers responds to multiple hormones

CnAIP2 (Callitropsis nootkatensis ABI3-Interacting Protein 2) was previously identified as a protein that interacts with the yellow-cedar ABI3 protein. CnAIP2 plays important roles during several key transitions of the plant lifecycle and acts as a global regulator with functions opposite to those of ABI3 proteins. Here we report that the CnAIP2 gene promoter is strongly upregulated by all of the major plant hormones. Young Arabidopsis seedlings expressing a chimeric CnAIP2_pro-GUS construct were subjected to exogenously applied hormones; the maximum fold-enhancement of GUS activity was as high as 47-fold, and each hormone showed a distinctive cell/tissue-specific pattern of GUS induction. By far the greatest response was elicited by the synthetic auxin 2,4-D (47-fold induction); the other hormones tested stimulated GUS activities by 8- to 21-fold. The CnAIP2 promoter also responded to glucose and salt (NaCl), albeit to a lesser extent (2- to 3-fold induction). As well as acting in an antagonistic way to the global regulator ABI3, CnAIP2 appears to participate in multiple hormonal crosstalk pathways to carry out its functions.     

A proposed sequence of hormones controlling the induction of luteal 20alpha-hydroxy steroid dehydrogenase and progesterone withdrawal in the late-pregnant rat.

1. The previously reported induction of luteal 20alpha-hydroxy steroid dehydrogenase by administration of aminoglutethimide to late-pregnant rats was shown to be unaffected by prior removal of the foetuses. Aminoglutethimide therefore does not act via the foetuses in this context. 2. The ability of injected oestrogen to prevent the above induction was lost by delaying the injection for 12h after aminoglutethimide, although the increase in enzyme activity begins only after 24h. 3. Induction of 20alpha-hydroxy steroid dehydrogenase by foetoplacental removal on day 18 of pregnancy was inhibited by human choriogonadotropin, lutropin (luteinizing hormone) and pregnant-mare serum gonadotropin, but not by somatotropin (growth hormone), thyrotropin or follitropin (follicle-stimulating hormone) 4. Indomethacin blocked the normal induction of 20alpha-hydroxy steroid dehydrogenase in late pregnancy and that caused by aminoglutethimide. It partially blocked that caused by human choriogonadotropin given on days 19-20 and that caused by 2-bromo-alpha-ergocryptine on days 5-6, but failed to block that caused by human choriogonadotropin on days 15-16 or by foetoplacental removal on day 18 of pregnancy. 5. These findings, and the control of progesterone synthesis in late pregnancy, are interpreted in terms of a sequence of hormonal or enzymic syntheses, each of which is inhibited by the product of the preceding synthesis.     

Induction of gamma-glutamyl transferase by dexamethasone in cultured fetal rat hepatocytes

Hepatocytes isolated as a relatively pure population from normal fetal rats were maintained in primary monolayer culture for 4-10 days. Hepatocytes exhibited a small increase in basal gamma-glutamyl transferase (GGT) activity over time. Exposure to dexamethasone (10(-6) mol/l) elicited a rise in GGT activity after a lag of 24 h. The presence of the steroid was necessary to maintain induction, and its removal resulted in reversal of induction. The maximal response was 2- to 3-fold, 72 h after exposure to the steroid. After this maximal response, a gradual decay in enzyme activity occurred, despite the continuous presence of the hormone. Actinomycin D or cycloheximide given prior to/or simultaneously with the steroid prevented the induction, thus suggesting that both RNA and protein biosynthesis are necessary for induction to occur.     

Turnover of endogenous, microinjected, and sequestered protein in Xenopus oocytes

Pulse-labeled oocyte proteins were found to have a maximum average half-life of 73 h. In general, larger peptides underwent degradation at a faster rate than smaller peptides. In this respect, oocytes are similar to most other cells. Microinjected ¹²⁵I-labeled bovine serum albumin (BSA) was degraded over a 40 h period with a half-life of 20–30 h, regardless of the method of protein labeling, culture medium employed, size of oocyte microinjected, or hormonal history of the oocyte. The last two results, if applicable to oocyte proteins in general, imply that protein catabolism is constant throughout the later stages of oogenesis and that growth is primarily regulated by a stimulation of anabolism. Individual proteins microinjected into oocytes undergo rates of degradation consistent with turnover rates obtained in other systems. Sequestered ¹²⁵I-labeled BSA is only partially (40%) degraded, which indicates that, unlike microinjected ¹²⁵I-labeled BSA, it has access to a cytoplasmic compartment (yolk platelets?) within which it is relatively stable.     

Nature of the thiamin-binding protein from chicken egg yolk.

A simple, rapid and efficient procedure for the purification of thiamin-binding protein from chicken egg yolk was developed. The method involved removal, by exclusion, of lipoproteins from DEAE-cellulose and subsequent elution of water-soluble proteins held on the ion-exchanger with 1 M-NaCl, followed by treatment of the eluted protein fraction with an aqueous suspension of dextran/charcoal to generate apoprotein from the holoprotein. The resultant protein fraction was subjected to bioaffinity chromatography on thiamin pyrophosphate--AE (aminoethyl)-Sepharose. The protein eluted specifically with 10 microM-thiamin at pH 7.0, was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, had a mol.wt. of 38 000 +/- 2000 and was not a glycoprotein. The purified thiamin-binding protein specifically interacted with riboflavin-binding protein with no detectable deleterious affect on its (14C)thiamin-binding capacity. The protein bound [14C]thiamin with a molar ratio of 1.0, with dissociation constant (Kd) 0.41 microM. This protein-ligand interaction was inhibited by thiamin analogues and antagonists. The absorption spectrum of the protein in the presence of thiamin exhibited significant hypochromism at the 278 nm band, indicating the involvement of aromatic amino acid residues of the protein, during its binding to the ligand. The protein cross-reacted with the monospecific antiserum to egg-white thiamin-binding protein, showing thereby that thiamin-binding proteins present in chicken egg yolk and white are the products of the same structural gene.     

Differential dynamics of receptor down-regulation and tyrosine aminotransferase induction following glucocorticoid treatment

Autoregulation of glucocorticoid receptor (GR) concentration in vivo may be an important determinant of steroid sensitivity. The dynamics of GR regulation were assessed and compared to regulation of tyrosine aminotransferase (TAT) expression in liver tissue taken from rats treated with a single 50 mg/kg i.v. dose of methylprednisolone. Plasma methylprednisolone concentrations were determined by HPLC analysis. Receptor and TAT message levels were determined by quantitative Northern hybridization. Methylprednisolone plasma kinetics showed a half-life of 0.6 h. Receptor occupancy occurred rapidly and cytosolic GR reappeared over 2–12 h. TAT activity rose between 2 and 6 h and then dissipated. Reduction in receptor mRNA levels occurred very rapidly, being detectable by 30 min following steroid administration. A down-regulated steady-state in GR message expression was reached by 2 h post-injection, and was maintained throughout the 18 h examined in this study. Comparison of methylprednisolone kinetics demonstrated that down-regulation was maintained long after drug was eliminated. In contrast, TAT message induction occurred with a sharp peak; maximal induction occurred between 5–6 h and return to baseline at approx. 8–10 h post-induction. This study shows that unlike TAT induction, GR message repression in vivo does not require continual presence of hormone.     

Ecdysterone-induced protein synthesis in vitro

Ecdysterone added in vitro to wing tissue from diapausing Antheraea polyphemus pupae induced the synthesis of several epidermal cell proteins. This is one of few instances in which any steroid hormone in physiological concentrations has been able to induce specific protein synthesis in target tissue in vitro soon after hormone stimulation. Hormone-treated tissue was incubated with ³H-leucine while control tissue was incubated with ¹⁴C-leucine. Polyacrylamide gel electrophoretic distribution of labelled wing tissue proteins after ecdysterone stimulation in vitro for various periods of time was determined. The ${}^3\text{H}{}^{14}\text{C}$ ratio emphasized the areas of increased protein synthesis due to ecdysterone. These areas of increased protein synthesis were reproducible with several ecdysterone concentrations and with different incubation times. Induction of protein synthesis occurs at an earlier time period when the hormone dosage is higher, i.e. the lower the dosage, the longer it is necessary for exposure of tissue to hormone. α-Ecdysone, known to initiate the moulting process in vitro in some insect species, also induced protein synthesis. Cortisol, a mammalian steroid hormone, produced no hormone specific protein synthesis. Therefore, the results seen with ecdysterone and α-ecdysone are not the result of non-specific steroid stimulation. When no hormone was added to the incubation medium (control), only one area of the polyacrylamide gel demonstrated protein synthesis. Therefore, there are a few proteins being synthesized in vitro in wing tissue, removed from diapausing animals without hormone stimulation, which may be related to the ‘injury phenomenon’. Protein banding patterns were also determined and compared with the radioactivity profile. The study of such early biochemical and physiological responses of target tissue to hormones will aid in our understanding of a hormone's mechanism of action, since the earlier an event occurs, the more likely that it is the primary result of hormone stimulation.     

A strategy for finding the optimal deconvolution estimates for hormone secretory kinetics using AutoDecon

AutoDecon is a fully automatic, multiparameter deconvolution procedure that can be used to estimate various hormone secretion kinetics. We propose a strategy based on the application of the corrected Akaike’s information criterion to select the optimal deconvolution model from a class of candidates generated by applying different combinations of initializing values. Using simulated cortisol time series, we show that, particularly in cases of diminished hormone half-life, this approach can yield estimates that are closer to the true underlying secretion kinetics compared with the commonly used approach based on a single set of initializing values. However, although we provide proof of principle, more extensive elaboration and validation of this approach are necessary.     

Osmium Zinc Iodide staining of Golgi elements in oocytes of Triturus cristatus

Summary Developing oocytes of the newt Triturus cristatus were studied in order to clarify the role played by the Golgi apparatus in the formation of yolk. The cytochemical method used for this purpose was that of Maillet (1968) which employs an Osmium Zinc Iodide (OZI) complex.Previtellogenic oocytes reveal a pattern of OZI staining only after hormonal (HCG) stimulation, following which both the Golgi apparatus and the multivesicular bodies are stained.Vitellogenic oocytes taken from non-hormonally stimulated females reveal OZI deposits in a number of vesicles peripheral to the Golgi apparatus as well as within the superficial layer of the forming yolk platelets. Following hormone stimulation, many of the Golgi apparatus located in the central ooplasm of vitellogenic oocytes have all their cisternae blackened by the OZI deposits; other apparatuses, more peripherally located, remain essentially unchanged in their staining pattern. Further, a large number of OZI stained vesicles becomes visible in the vicinity of the Golgi apparatus and within the superficial layer of the forming yolk platelets.The present findings are interpreted as indicating the occurrence of fusion between Golgi derived vesicles and forming yolk platelets. It is also suggested that the vesicles in question function as carriers of Golgi produced enzymes which are presumably required to accomplish the final elaboration of the yolk material.Supported by a grant from the Consiglio Nazionale delle RicercheWe acknowledge the valuable help received from Prof. G. Mancino throughout this investigation     

The regulation of ornithine aminotransferase synthesis by glucagon in the rat.

Ornithine aminotransferase (OAT) from rat liver mitochondria was purified to homogeneity. A monospecific antiserum against the enzyme protein was prepared in rabbits. Immunotitrations were performed on OAT present in crude mitochondrial extracts obtained from the livers and kidneys of rats in several hormonal and dietary states. No evidence was found for the existence of an immunologically reactive but enzymatically inactive form of OAT. The relative rate of enzyme synthesis in vivo was studied by pulselabeling rats with [4, 5-³H]leucine, isolating the enzyme protein by immunoprecipitation, and dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels. Nine hours after a single subcutaneous injection of a glucagon oil emulsion, a 3-fold increase in OAT activity and a 12-fold increase in the synthetic rate of the enzyme were observed. Serine dehydratase activity increased on a time-course very similar to that of OAT following glucagon injection. These increases occurred only on low (0–12.5%) protein diets. At higher levels of dietary protein (30% and up), no further stimulation of OAT synthesis by glucagon was observed. Administration of actinomycin D within the first 2 h after glucagon injection resulted in an inhibition of OAT induction. When the administration of the antibiotic was delayed until 4 h after glucagon, no inhibition of OAT induction was observed. Glucose repression of the glucagon induction of the enzyme in hepatic mitochondria was demonstrated to be the result of a rapid inhibition of OAT synthesis.     

Regulation of ovarian ornithine decarboxylase: Role of calcium ions in enzyme induction in isolated swine granulosa cells in vitro

When swine granulosa cells were cultured in chemically defined medium selectively deficient in Ca²⁺, the dose-dependent stimulation of ornithine decarboxylase (EC 4.1.1.17) activity in response to prostaglandin E₂, l-epinephrine or the somatomedin, multiplication-stimulating activity, was attenuated markedly. Putative calcium influx blockers, verapamil and diltiazem, also inhibited hormone-stimulated enzymic activity. Similar inhibitory effects were exerted by divalent (cobalt) or trivalent (lanthanum) cations believed to compete with calcium for extracellular binding sites. The suppressive effects of extracellular calcium deprivation were time-dependent (suggesting gradual depletion of intracellular calcium stores), and could be mimicked by the intracellular antagonist of calcium action, trifluoperazine. The mechanism(s) subserving diminished hormonal induction of enzyme activity could not be accounted for by alterations in cell viability, general protein synthesis, half-life of decay of enzyme activity (measured in the presence of cycloheximide), or apparent K_m of ornithine decarboxylase. Ca²⁺ and/or calcium antagonists did not modify enzyme activity in cell-free preparations. These observations implicate Ca²⁺ in the hormonal induction of a discrete cytosolic enzyme in isolated intact ovarian cells.