Synthesis of DNA fragments containing 2'-deoxy-4'-selenonucleoside units using DNA polymerases: comparison of dNTPs with O, S and Se at the 4'-position in replication.

4'-SelenoDNA fragments were synthesized for the first time using 4'-selenothymidine triphosphate (SeTTP) by taking advantage of its bioequivalence against DNA polymerases. DNA fragments each with a homologous element (O, S or Se) at the 4'-position of the thymidine units were effectively amplified using KOD Dash DNA polymerase.

Bactericidal activity and oral pathogen inactivation by electromagnetic wave irradiation.

AIMS: The aim of this work was to clarify the effects of electromagnetic wave irradiation (EMWI) on oral bacterial pathogens. METHODS AND RESULTS: A Gram-negative (Porphyromonas gingivalis) or Gram-positive (Streptococcus mutans, S. intermedius, Enterococcus faecalis) bacterial suspension was irradiated by EMW apparatus (500-1000 kHz, 5-15 times, 1 s time(-1) ). Quantification of survival bacteria by CFU counting revealed that EMWI exhibited marked bactericidal activity against all tested bacteria and bactericidal activity at 500 kHz increased in an irradiation number-dependent manner. After EMWI at 500 kHz, scanning electron microscopic observations showed that the chain of S. mutans cells was shortened after 5 irradiations and the outlines of bacterial cells (S. mutans and P. gingivalis) were unclear after 5-10 irradiations. EMWI inhibited the inductive effect of S. mutans on pro-inflammatory cytokine production in human monocytes and this inhibitory effect was comparable with that of heat-killed bacteria. Furthermore, using an enzyme activity assay, EMWI partially inactivated the activities of gingipains from P. gingivalis. CONCLUSIONS: These findings demonstrated that EMWI has inactivation and bactericidal activities against single microbial species among four kinds of oral pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Electromagnetic wave irradiation may be applicable for medical disinfection and sterilization, such as refractory periapical periodontitis.

Saireito (a Chinese herbal drug)-stimulated secretion and synthesis of pituitary ACTH are mediated by hypothalamic corticotropin-releasing factor.

Administration of Saireito, a Saiko agent (a Chinese herbal drug), via a stomach cannula stimulates ACTH release and proopiomelanocortin, the precursor for ACTH, gene expression in the rat anterior pituitary. To study whether Saireito-stimulated secretion and synthesis of ACTH are mediated by hypothalamic corticotropin-releasing factor (CRF), we examined the effect of passive immunization of endogenous CRF by i.v. administration of CRF antiserum on Saireito-increased plasma ACTH levels and proopiomelanocortin gene expression in the rat anterior pituitary, under pentobarbital anesthesia. CRF antiserum inhibited Saireito-induced plasma ACTH levels and proopiomelanocortin mRNA levels in the anterior pituitary. This result indicates that Saireito stimulates CRF neurons to increase CRF release, which stimulates secretion and synthesis of ACTH.

Stimulatory effect of Saireito on proopiomelanocortin gene expression in the rat anterior pituitary gland.

The effect of administration of Saireito, a Saiko agent, via a stomach cannula on adrenocorticotropin (ACTH) release and gene expression of proopiomelanocortin (POMC), the precursor for ACTH, in the anterior pituitary, as well as on the corticotropin-releasing factor (CRF) in the hypothalamus, was examined in pentobarbital anesthetized rats. Saireito decreased the hypothalamic CRF level due to an early release of CRF and stimulated ACTH release and POMC gene expression but did not increase CRF gene expression. These results suggest that Saireito does not stimulate CRF gene expression, although it does stimulate CRF release, which in turn stimulates POMC gene expression in the anterior pituitary and ACTH release.

Neuropeptide Y increases the corticotropin-releasing factor messenger ribonucleic acid level in the rat hypothalamus.

Neuropeptide Y (NPY) has a stimulatory effect on adrenocorticotropin (ACTH) and corticotropin-releasing factor (CRF) release. In the present study, to investigate the effect of NPY on CRF synthesis, the effect of centrally administered NPY on CRF messenger RNA (mRNA) levels in rat hypothalamus was examined under pentobarbital anesthesia. The administration of 0.01, 0.1 and 1 nmol of NPY into the lateral ventricle dose-dependently Increased the plasma ACTH levels, as well as the levels of proopiomelanocortin mRNA in the anterior pituitary. The CRF mRNA level in the hypothalamus also increased after administration of 0.1 and 1 nmol of NPY in a dose-dependent manner. The administration of 3 nmol of phentolamine or propranolol failed to block 0.1 nmol NPY-induced ACTH release or 1 nmol NPY-stimulated CRF mRNA levels in the hypothalamus. These results Indicate that the central administration of NPY increases the CRF mRNA levels in the hypothalamus and the probable CRF release, which increases the proopiomelanocortin mRNA levels and ACTH secretion in the anterior pituitary. Therefore, NPY seems to play a physiological role in the regulation of the release and synthesis of CRF in the hypothalamus.

The role of corticotropin-releasing factor and vasopressin in hypoglycemia-induced proopiomelanocortin gene expression in the rat anterior pituitary gland.

In this study, we examined the effect of passive immunization of endogenous corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) on hypoglycemia-induced adrenocorticotropic hormone (ACTH) secretion and determined proopiomelanocortin messenger RNA (POMC mRNA) levels in the anterior pituitary as well as hypothalamic CRF mRNA levels in pentobarbital anesthetized rats. The response of plasma ACTH to hypoglycemia was partially inhibited by the administration of CRF-antiserum (CRF-As) or AVP-antiserum (AVP-As) alone, but was found to be completely abolished by the administration of CRF-As + AVP-As as compared to the response in normal rabbit serum-treated rats. The hypoglycemia-induced POMC mRNA level in the anterior pituitary was completely inhibited by the administration of CRF-As alone and CRF-As + AVP-As, but was not inhibited by AVP-As alone as compared to the response in normal rabbit serum-treated rats. The administration of CRF-As and/or AVP-As did not affect hypoglycemia-induced CRF mRNA levels in the hypothalamus. These results indicate that the synergistic effect of CRF and AVP is important for hypoglycemia-induced ACTH secretion, but CRF is essential and indispensable for hypoglycemia-induced POMC gene expression in the anterior pituitary (AP).

Beta-endorphin inhibits hypoglycemia-induced gene expression of corticotropin-releasing factor in the rat hypothalamus.

Endogenous opioid peptides have a role in the regulation of the hypothalamic-pituitary-adrenal axis. Recently, beta-endorphin (EP) has been thought to inhibit CRF release in vivo and in vitro. In the present study we examined the effects of central administration of EP on ACTH secretion and gene expression of both CRF in the hypothalamus and POMC in the anterior pituitary gland (AP) during basal and insulin-induced hypoglycemia in pentobarbital-anesthetized rats. Administration of EP in the lateral ventricle decreased basal CRF levels in the median eminence and inhibited basal and hypoglycemia-induced ACTH secretion in a dose-dependent manner. Hypoglycemia-induced POMC mRNA levels in the AP and CRF mRNA levels in the hypothalamus were also dose-dependently inhibited by the administration of EP. The inhibitory effect of EP was reversed by naloxone. These results suggest that 1) central administration of EP acts through the opioid receptor to inhibit hypoglycemia-induced CRF gene expression in the hypothalamus and CRF release, which results in a decrease in ACTH secretion and POMC mRNA levels in the AP; and 2) the active site of EP is the CRF neuron in the paraventricular nucleus.

Hypothalamic corticotropin-releasing hormone (CRH) secretion into hypophysial portal blood is regulated by cutaneous sensory stimulation in anesthetized rats.

The effects of noxious and non-noxious mechanical stimulation of various segmental skin areas (face, forelimb and forepaw, abdomen, hindlimb and hindpaw) on the secretion of immunoreactive corticotropin-releasing hormone (iCRH) from the hypothalamus into hypophysial portal blood was examined in artificially ventilated rats under halothane anesthesia. Secretion of iCRH was calculated from the iCRH concentration in hypophysial portal plasma and the plasma flow rate. Noxious mechanical stimulation of the skin was delivered by pinching using surgical clamps, while non-noxious mechanical stimulation was provided by brushing with tooth brushes. Pinching of the bilateral forepaws or hindpaws and brushing of the bilateral hindlimbs for 20 min increased hypothalamic iCRH secretion. In contrast, pinching of the face or abdomen and brushing of the face, forelimbs, or abdomen for 20 min did not significantly influence it. These results indicate that cutaneous mechanical sensory stimulation contributes to the reflex regulation of CRH secretion from the hypothalamus into hypophysial portal blood, and also that this effect is highly dependent on the site of stimulation.

Presence of CRH-binding protein in amniotic fluid and in umbilical cord plasma.

CRH-binding protein was present in the amniotic fluid and in the umbilical cord plasma after 15 weeks and 24 weeks of pregnancy, respectively. The size of the CRH-binding protein was similar to that in the peripheral blood from normal subjects. The level of the binding of CRH-binding protein in the umbilical cord plasma during the third trimester of pregnancy was also similar to that in the peripheral blood of neonates and normal adult subjects. The binding of CRH-binding protein was temporarily decreased at 40 weeks of pregnancy. These results indicate that fetal CRH-binding protein seems to be produced at least in the second trimester of pregnancy.

Effects of sex steroids on beta-endorphin release from rat hypothalamus in vitro.

The effects of sex steroids on immunoreactive beta-endorphin (EP) release from the rat hypothalamus in vitro were examined using a rat hypothalamic perifusion system and an EP RIA. Testosterone (1-100 ng/ml) and estradiol (10-100 pg/ml) stimulated EP release in a dose-dependent manner. Dehydroepiandrosterone (DHEA, 1-100 ng/ml) dose-dependently inhibited EP release. These results indicate an inverse relationship between the acute effect of gonadal sex steroids and that of adrenal androgen on hypothalamic EP release in vitro.

Angiotensin II increases the corticotropin-releasing factor messenger ribonucleic acid level in the rat hypothalamus.

Angiotensin II (AII) has an important role in the regulation of CRF release. In the present study, the effect of centrally administered AII on CRF messenger RNA (mRNA) levels in the rat hypothalamus was examined. Administration of 0.1 nmol and 1 nmol AII into the lateral ventricle increased the levels of plasma ACTH 20 min and 45 min after administration and those of proopiomelanocortin mRNA in the anterior pituitary (AP) and CRF mRNA in the hypothalamus 2 h after administration. On the other hand, ACTH levels in AP and CRF levels in the median eminence temporarily decreased 45 min after the administration of 1 nmol AII, but it returned to the control level at 90 min. Administration of 10 nmol saralacin, an AII antagonist, blocked 1 nmol AII-induced increase in the levels of plasma ACTH, proopiomelanocortin mRNA in AP, and CRF mRNA in the hypothalamus. These results indicate that central administration of AII increases the CRF mRNA level in the hypothalamus in a receptor-specific manner and also increases CRF release. Therefore, AII seems to have an important role in the regulation of the release and synthesis of CRF in the hypothalamus.

Glucocorticoids decrease a binding of corticotropin-releasing hormone-binding protein in human plasma.

The binding of CRH-binding protein (CRH-BP) in plasma to labeled human CRH has been examined in patients with hypothalamic-pituitary-adrenal disorders. Compared with that in normal subjects, CRH-BP binding decreased in patients with Cushing's syndrome of pituitary or adrenal origin and in patients who were treated with a high dose of glucocorticoids over a long period of time. On the other hand, CRH-BP binding increased in patients with Addison's disease or hypopituitarism. In patients with Addison's disease, the high level of CRH-BP binding fell to the control level after glucocorticoid replacement. In patients with Cushing's syndrome, CRH-BP binding gradually increased and reached the higher level about 1 yr after surgery. Thereafter, it returned to the control level. There was a good negative correlation between the levels of plasma cortisol and CRH-BP binding in patients with Cushing's syndrome before and after surgery. A Scatchard analysis of CRH-BP binding in patients with Cushing's syndrome and in normal subjects showed that the binding affinity was similar in both groups, but that the number of binding sites was low in patients with Cushing's syndrome. These results suggest that in human plasma, glucocorticoids decrease CRH-BP binding. This seems to be caused by a decrease in the concentration of CRH-BP in the plasma of patients with hypercortisolemia.

Interleukin-1 stimulates corticotropin-releasing factor gene expression in rat hypothalamus.

To examine the effect of interleukin-1 (IL-1) on CRF and POMC gene expression, recombinant human IL-1 alpha and -beta were ip injected in rats. The plasma ACTH level showed a dose-related increase at 2 h after the injection of 0.5 and 2 micrograms IL-1 alpha and -beta, and also showed a sustained increase from 1 h until 5 h after the injection of 2 micrograms of IL-1 beta. CRF contents in the medial basal hypothalamus and ACTH contents in the anterior pituitary (AP) decreased at 2 h after the injection of 2 micrograms of IL-1 alpha and -beta, and such decreased levels were maintained until 5 h after the injection of 2 micrograms of IL-1 beta. The levels of CRF mRNA in the hypothalamus and POMC mRNA in AP significantly increased 3 h after the injection of 2 micrograms IL-1 alpha and -beta, and these levels were still higher at 5 h after the injection of 2 micrograms of IL-1 beta compared with those of the control. There was no significant change in the ACTH content and POMC mRNA levels in the intermediate-posterior pituitary or the hypothalamus or in the CRF contents and CRF mRNA levels in the cerebral cortex. These results indicate that acute administration of IL-1 alpha and -beta stimulates gene expression of hypothalamic CRF and CRF release, which causes the stimulation of ACTH release and POMC gene expression in AP.

Corticotropin-releasing factor-binding protein is a glycoprotein.

Human corticotropin-releasing factor-binding protein (hCRF-BP), a 38,000 dalton protein, specifically binds hCRF in plasma. CRF-BP-CRF complex adsorbed to concanavalin-A-Sepharose and its Mr decreased after treatment with endoglycosidase H or glycopeptidase A. The binding of CRF-BP to CRF decreased after treatment with endoglycosidase H. These results indicate that the CRF-BP is a glycoprotein that contains asparagine N-linked-type oligosaccharides, and such oligosaccharide chains are important for CRF-BP binding.

Responses to corticotropin-releasing hormone and its bound and free forms in pregnant and nonpregnant women.

Plasma CRH levels are considerably higher in women during the third trimester of pregnancy than in non-pregnant women. Most of plasma CRH in pregnant women is bound to CRH-binding protein (CRH-BP). To gain further insight into CRH physiology during pregnancy, we measured the responses of plasma ACTH and cortisol and the changes in bound and free forms of CRH in plasma after human CRH administration (2 micrograms/kg) in five pregnant (39-40 weeks of pregnancy) and five nonpregnant women. The mean basal plasma ACTH and cortisol levels in the pregnant women were higher than those in the nonpregnant women. However, the maximum increments in plasma ACTH and cortisol levels and the integrated ACTH and cortisol responses, after subtraction of the basal levels after CRH administration, were similar in the two groups. The plasma CRH half-time in the pregnant group was similar to that in the nonpregnant group. The mean basal plasma CRH level in the nonpregnant women was 1.5 +/- 0.2 (+/- SE) pmol/L, and that in the pregnant women was 360 +/- 35 pmol/L. On gel filtration chromatography, almost all of the CRH in the plasma was protein bound (320 +/- 30 pmol/L) in the pregnant women; no CRH peaks were detected in nonpregnant women because of the low plasma CRH levels. After CRH administration, the level of the bound form of plasma CRH was highest at 5 min, and then declined to a plateau at 15 min and 30 min in the pregnant women. In the nonpregnant women, protein-bound CRH also was highest at 5 min, but it progressively declined thereafter. The disappearance rate of the bound CRH in plasma from the nonpregnant women was similar to that of the second compartment of the plasma decay curves of the free CRH from both groups. We conclude that the plasma ACTH and cortisol responses to exogenous CRH are similar in pregnant and nonpregnant women, the effect of CRH-BP on the disappearance of plasma CRH is minimal, and plasma CRH-BP in pregnant women has the capacity to bind additional CRH.

Effects of protein kinase-C-related adrenocorticotropin secretagogues and interleukin-1 on proopiomelanocortin gene expression in rat anterior pituitary cells.

To examine the effects of the cAMP-independent protein kinase-C system and interleukin-1 (IL-1) on secretion of ACTH and POMC gene expression in cultured rat anterior pituitary (AP) cells, AP cells were incubated with CRF, 8-bromo-cAMP, arginine vasopressin, angiotensin II, norepinephrine, and phorbol 12-myristate 13-acetate. After 15 h of incubation, CRF and 8-bromo-cAMP increased both ACTH release and the POMC mRNA level. Arginine vasopressin, angiotensin II, norepinephrine, or phorbol 12-myristate 13-acetate stimulated ACTH release but failed to increase basal or CRF-stimulated POMC mRNA levels. Human recombinant IL-1 alpha and -beta increased neither ACTH release nor POMC mRNA levels after 3 h of incubation. After 15 h of incubation, 100 pM to 10 nM IL-1 alpha and -beta increased ACTH release. However, POMC mRNA levels were significantly elevated only by 10 pM IL-1 beta. These results suggest that the CRF-cAMP system plays a major role in both ACTH release and expression of the POMC gene in AP cells, but the cAMP-independent protein kinase-C system contributes only to ACTH release; that acute stimulation of ACTH release from AP with IL-1 administration is not due to direct action of IL-1 at the pituitary level; that chronic exposure of AP cells to IL-1 alpha or -beta can stimulate ACTH release; and that the direct effects of IL-1 alpha and -beta on POMC gene expression, if any, seem to be minimal.

Adrenergic modulation of adrenocorticotropin responses to insulin-induced hypoglycemia and corticotropin-releasing hormone.

To study possible adrenergic modulation of pituitary-adrenal responses to insulin-induced hypoglycemia and CRH we examined the effect of nonselective alpha-blockade (phentolamine) and nonselective beta-blockade (propranolol) on plasma ACTH, cortisol, and vasopressin (AVP) responses to hypoglycemia and CRH in five normal men. Infusion of propranolol or phentolamine did not alter basal plasma ACTH or cortisol levels. The propranolol infusion enhanced the stimulatory effect of hypoglycemia on ACTH, cortisol, and AVP secretion and also enhanced the stimulatory effect of CRH on ACTH and cortisol secretion. Infusion of phentolamine inhibited hypoglycemia-induced ACTH and AVP secretion, but had no effect on the stimulatory effect of CRH on ACTH and cortisol secretion. The increments of plasma ACTH and cortisol induced by an almost maximal dose of CRH (1 microgram/kg) were smaller than those induced by hypoglycemia. The propranolol-induced enhancement of the ACTH response to hypoglycemia was almost the same as the ACTH response to CRH alone. From these results we conclude that propranolol may act at the pituitary level to enhance CRH action, rather than AVP action, and that the ACTH response to hypoglycemia may be mediated by hypothalamic alpha-adrenergic activation.

Characterization of corticotropin-releasing hormone binding protein in human plasma by chemical cross-linking and its binding during pregnancy.

A human plasma CRH-binding protein (CRH-BP) was identified and characterized by chemical cross-linking of 125I-Tyr-hCRH to human plasma using disuccinimidyl suberate. The apparent mol wt of the cross-linked complex determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography was approximately 43,000. The mol wt was slightly lower in the nonreduced state, suggesting the presence of intramolecular disulfide bonds. Subtracting the mol wt of 125I-Tyr-CRH, the BP appeared to have a mol wt of approximately 38,000. Binding was specific since the appearance of the 43,000 dalton band was not affected by unlabeled ACTH, vasopressin, serum albumin, or gamma-globulin, but was inhibited by unlabeled hCRH dose dependently. Pretreatment of plasma with 0.1 mol/L HCl, 0.01 mol/L NaOH, 10 mmol/L dithiothreitol, or trypsin before cross-linking abolished its ability to bind 125I-Tyr-hCRH. Rat, rabbit, or goat plasma or human cerebrospinal fluid did not bind 125I-Tyr-CRH. It is unlikely that CRH-BP is a CRH receptor, because the estimated mol wt of the CRH-BP is smaller than the reported size of CRH receptors, and the CRH-BP did not bind to ovine CRH. The binding of 125I-Tyr-CRH to CRH-BP decreased in the third trimester of pregnancy, when plasma CRH levels were markedly elevated. However, after dissociating endogenous CRH from the CRH-BP, the binding was almost the same as in nonpregnant subjects. In addition, CRH-BP inhibited CRH-induced ACTH secretion from cultured rat anterior pituitary cells. We conclude that most of the increased plasma CRH found in pregnant women is bound to CRH-BP, and so is inactive, therefore plasma ACTH levels do not increase to above the normal range.

Insulin-induced hypoglycemia increases corticotropin-releasing factor messenger ribonucleic acid levels in rat hypothalamus.

To study the effect of acute stress on CRF release and synthesis in rat hypothalamus, ACTH levels in plasma, CRF contents in the median eminence (ME), and CRF mRNA levels in the hypothalamus without ME and cerebral cortex were determined after insulin-induced hypoglycemia. Plasma ACTH levels increased at 30 and 60 min, while ME CRF content decreased at 30 and 60 min, then returned to the control level at 90 min. Hybridization with a cRNA probe revealed a single size class of CRF mRNA in the hypothalamus and cerebral cortex (approximately 1300 nucleotides), and the size of CRF mRNA in these tissues did not change during the experimental period. CRF mRNA levels in the hypothalamus increased to 130% of the control value at 30 min and reached a peak (186% of the control value) at 120 min, but these levels in the cerebral cortex did not change. These results suggest that insulin-induced hypoglycemia stimulates CRF synthesis by increasing CRF mRNA levels in the hypothalamus as well as CRF release, and that release and synthesis of CRF in the cerebral cortex are independent of those in the hypothalamus.

Effects of corticotropin-releasing hormone and dexamethasone on proopiomelanocortin messenger RNA level in human corticotroph adenoma cells in vitro.

The effects of corticotropin-releasing hormone (CRH) and dexamethasone on proopiomelanocortin (POMC) mRNA levels in cultured pituitary adenoma cells were studied in 10 patients with Cushing's disease. As a control, POMC mRNA levels in cells from nonadenomatous tissues were examined in four patients. Human POMC mRNA in the cells was analyzed by Northern blot hybridization. Human POMC DNA probe hybridized with only a single size class of RNA (approximately 1,200 nucleotides) from the adenoma and nonadenoma cells of each patient. The size of POMC mRNA did not change through the culture or after incubation with CRH or dexamethasone. CRH increased POMC mRNA levels in these cells in a dose- and time-dependent manner. The minimum concentration of CRH required to elevate POMC mRNA levels in these cells exposed for 15 h was 0.1 nM. The minimum duration of 1 nM CRH treatment required to increase these levels was 3 h under our conditions. Inhibitory effects of 1 and 10 micrograms/dl dexamethasone on ACTH release and POMC mRNA levels in nonadenoma cells were greater than those in adenoma cells. These results suggest the following: (a) that the mRNA in cultured pituitary adenoma cells is qualitatively the same as that in vivo; (b) that responses of mRNA levels to CRH are time- and dose-dependent; and (c) that adenoma cells resist the inhibitory effect of dexamethasone on POMC mRNA levels and ACTH release.