N1s and O1s double ionization of the NO and N2O molecules.

Single-site N1s and O1s double core ionisation of the NO and N2O molecules has been studied using a magnetic bottle many-electron coincidence time-of-flight spectrometer at photon energies of 1100 eV and 1300 eV. The double core hole energies obtained for NO are 904.8 eV (N1s(-2)) and 1179.4 eV (O1s(-2)). The corresponding energies obtained for N2O are 896.9 eV (terminal N1s(-2)), 906.5 eV (central N1s(-2)), and 1174.1 eV (O1s(-2)). The ratio between the double and single ionisation energies are in all cases close or equal to 2.20. Large chemical shifts are observed in some cases which suggest that reorganisation of the electrons upon the double ionization is significant. Δ-self-consistent field and complete active space self-consistent field (CASSCF) calculations were performed for both molecules and they are in good agreement with these results. Auger spectra of N2O, associated with the decay of the terminal and central N1s(-2) as well as with the O1s(-2) dicationic states, were extracted showing the two electrons emitted as a result of filling the double core holes. The spectra, which are interpreted using CASSCF and complete active space configuration interaction calculations, show atomic-like character. The cross section ratio between double and single core hole creation was estimated as 1.6 × 10(-3) for nitrogen at 1100 eV and as 1.3 × 10(-3) for oxygen at 1300 eV.

Single and multiple photoionisation of H2S by 40-250 eV photons.

Multi-electron coincidence measurements on photoionisation of H(2)S have been carried out at photon energies from 40 to 250 eV. They quantify molecular field effects on the Auger process in detail and are in good agreement with the existing theory. Spectra of core-valence double ionisation of H(2)S are presented and partially analysed. Auger decays from the core-valence states produce triply charged product spectra with unexplained and surprising intensity distributions. Triple ionisation by the double Auger process from 2p hole states shows little effect of the molecular field splitting, but includes a substantial contribution from cascade processes, some involving dissociation in intermediate states. The onset of triple ionisation at the molecular geometry is determined as 61 ± 0.5 eV.

Triple ionization of CO2 by valence and inner shell photoionization.

Spectra of triply ionized CO(2) have been obtained from photoionization of the molecule using soft x-ray synchrotron light and an efficient multi-electron coincidence technique. Although all states of the CO(2) (+++) trication are unstable, the ionization energy for formation of molecular ions at a geometry similar to that of the neutral molecule is determined as 74 ± 0.5 eV.

Triple ionization spectra by coincidence measurements of double Auger decay: The case of OCS.

By combining multiple electron coincidence detection with ionization by synchrotron radiation, we have obtained resolved spectra of the OCS(3+) ion created through the double Auger effect. The form of the spectra depends critically on the identity of the atom bearing the initial hole. High and intermediate level electron structure calculations lead to an assignment of the resolved spectrum from ionization via the S 2p hole. From the analysis it appears that the double Auger effect from closed shell molecules favors formation of doublet states over quartet states. Molecular field effects in the double Auger effect are similar to those in the single Auger effect in linear molecules.

Core-valence double photoionization of the CS(2) molecule.

Double photoionization spectra of the CS(2) molecule have been recorded using the TOF-PEPECO technique in combination with synchrotron radiation at the photon energies hν=220, 230, 240, 243, and 362.7 eV. The spectra were recorded in the S 2p and C 1s inner-shell ionization regions and reflect dicationic states formed out of one inner-shell vacancy and one vacancy in the valence region. MCSCF calculations were performed to model the energies of the dicationic states. The spectra associated with a S 2p vacancy are well structured and have been interpreted in some detail by comparison to conventional S 2p and valence photoelectron spectra. The lowest inner-shell-valence dicationic state is observed at the vertical double ionization energy 188.45 eV and is associated with a (2p(3/2))(-1)(2π(g))(-1) double vacancy. The spectrum connected to the C 1s vacancy shows a distinct line at 310.8 eV, accompanied by additional broad features at higher double ionization energies. This line is associated with a (C 1s)(-1)(2π(g))(-1) double vacancy.

Spectra of the triply charged ion CS(2)(3+) and selectivity in molecular Auger effects.

Spectra of triply charged carbon disulphide have been obtained by measuring, in coincidence, all three electrons ejected in its formation by photoionization. Measurements of the CS(2)(3+) ion in coincidence with the three electrons identify the energy range where stable trications are formed. A sharp peak in this energy range is identified as the (2)Pi ground state at 53.1+/-0.1 eV, which is the lowest electronic state according to ab initio molecular orbital calculations. Triple ionization by the double Auger effect is provisionally divided, on the basis of the pattern of energy sharing between the two Auger electrons into contributions from direct and cascade Auger processes. The spectra from the direct double Auger effect via S 2p, S 2s, and C 1s hole states contain several resolved features and show selectivity based on the initial charge localization and on the identity of the initial state. Triple ionization spectra from single Auger decay of S 2p-based core-valence states CS(2)(2+) show retention of the valence holes in this Auger process. Related ion-electron coincidence measurements give the triple ionization yields and the breakdown patterns in triple photoionization at selected photon energies from 90 eV to above the inner shell edges.

Bendamustine, etoposide and dexamethasone to mobilize peripheral blood hematopoietic stem cells for autologous transplantation in patients with multiple myeloma.

Chemotherapeutic agents without cross-resistance to prior therapies may enhance PBSC collection and improve patient outcomes by exacting a more potent direct antitumor effect before autologous stem cell transplant. Bendamustine has broad clinical activity in transplantable lymphoid malignancies, but concern remains over the potential adverse impact of this combined alkylator-nucleoside analog on stem cell mobilization. We performed a prospective, nonrandomized phase II study including 34 patients with multiple myeloma (MM) (n=34; International Staging System (ISS) stages I (35%), II (29%) and III (24%); not scored (13%)) to evaluate bendamustine's efficacy and safety as a stem cell mobilizing agent. Patients received bendamustine (120 mg/m2 IV days 1, 2), etoposide (200 mg/m2 IV days 1-3) and dexamethasone (40 mg PO days 1- 4) (bendamustine, etoposide and dexamethasone (BED)) followed by filgrastim (10 µg/kg/day SC; through collection). All patients (100%) successfully yielded stem cells (median of 21.60 × 106/kg of body weight; range 9.24-55.5 × 106/kg), and 88% required a single apheresis. Six nonhematologic serious adverse events were observed in 6 patients including: neutropenic fever (1, grade 3), bone pain (1, grade 3) and renal insufficiency (1, grade 1). In conclusion, BED safely and effectively mobilizes hematopoietic stem cells.

Expression and hormonal regulation of the CCAAT enhancer binding protein-alpha during differentiation of rat ovarian follicles.

Follicular development involves both proliferation and differentiation of thecal and granulosa cells. The process is regulated by gonadotropins and paracrine and autocrine factors, including steroid hormones, presumably by the induction of different genes at specific time points. In the present study, the expression and distribution of the CCAAT enhancer-binding protein-alpha (C/EBP alpha) were studied in immature ovaries and in ovaries in which follicular growth and development were initiated with PMSG, whereas ovulation and luteal formation were induced by the injection of hCG. Ovaries were collected before and at different time points after PMSG (0, 6, 24, and 48 h) and hCG (0.25, 1, 3, 10, and 24 h) treatment for analyses of the contents of C/EBP alpha mRNA and protein and the cell-specific immunohistochemical localization of the protein. C/EBP alpha mRNA increased to maximal levels 24 h after PMSG treatment. The effect was specific for the ovary, as C/EBP alpha mRNA in the uterus did not change. C/EBP alpha mRNA decreased 10 h after hCG treatment and increased again in newly formed corpora lutea. Immunohistochemistry and immunoblotting demonstrated a similar increase in C/EBP alpha during follicular development. To examine the involvement of specific hormones in the regulation of C/EBP alpha, hypophysectomized immature rats were injected sequentially with estradiol and FSH. This treatment resulted in a substantial increase in C/EBP alpha mRNA and protein. These results demonstrate that C/EBP alpha is hormonally regulated in the ovary and suggest a role for C/EBP alpha during differentiation of ovarian cells and follicular development.

Differentiation of rat ovarian thecal cells: evidence for functional luteinization.

To determine if thecal cells of rat preovulatory (PO) follicles become functionally luteinized, theca from small antral (SA) and PO follicles were isolated before and 8 h after iv injection of an ovulatory dose (10 IU) of hCG. Thecal explants were cultured for 30 days in Dulbecco's Modified Eagle's Medium-Ham's F-12 medium containing 1% fetal calf serum (FCS) with or without 5 ng/ml ovine LH or 10 microM forskolin. Whereas theca from SA, hCG-treated SA, and PO follicles were dependent on LH or forskolin to maintain progesterone (greater than 10 ng/ml) and androstenedione (greater than 10 ng/ml) accumulation, luteinizing theca (hCG-treated PO) accumulated more than 10 ng/ml progesterone and more than 2 ng/ml androstenedione with or without LH or forskolin for 30 days. Granulosa cells were isolated from these same follicles and cultured under similar conditions, including 10 ng/ml testosterone and 25 ng/ml ovine FSH. Only granulosa cells isolated from luteinizing follicles (hCG-treated PO) maintained progesterone (greater than 20 ng/ml) and estradiol (10 ng/ml) accumulation with or without FSH or forskolin for 30 days. Basal concentrations of cAMP were 5 to 10-fold higher in thecal and granulosa cells from luteinizing follicles than in these tissues isolated from SA or PO follicles. We conclude that thecal cells as well as granulosa cells of rat PO follicles respond to the LH/hCG surge by becoming functionally luteinized, less dependent on LH, and capable of maintaining an increased accumulation of basal cAMP. Furthermore, the data suggest that one luteinizing thecal explant produces a similar amount of progesterone as one follicle equivalent of luteinizing granulosa cells. Thus, luteinized theca have the potential of contributing significantly to progesterone secretion by the mature rat corpus luteum.

The role of cyclic adenosine 3',5'-monophosphate in the ovulatory process of the in vitro perfused rabbit ovary.

LH alters ovarian steroidogenesis via adenylate cyclase (AC) activation and cAMP production. Although LH also initiates ovarian follicle rupture, evidence is lacking for involvement of cAMP in this process. This work explores the involvement of cAMP in the ovulation of in vitro perfused rabbit ovaries by comparing LH stimulation of ovaries with that of LH plus 3-isobutyl-1-methyl-xanthine (IBMX), (an inhibitor of phosphodiesterase) and of forskolin (a nonreceptor-specific activator of AC). Venous perfusates were analyzed for cAMP, progesterone, 17 beta-estradiol, and testosterone, ovaries were analyzed for cAMP, and ovulations were noted. LH, LH plus IBMX, and forskolin all increased tissue cAMP levels significantly after 0.5 h, the perfusate levels increasing rapidly thereafter reaching plateau levels, while tissue levels returned to control levels after 2.4 h. LH plus IBMX and forskolin significantly increased cAMP release over LH controls, LH plus IBMX increasing and forskolin decreasing the number of ovulations. Forskolin significantly increased progesterone release over LH controls and, although no other significant steroid differences were seen, strong tendencies existed. Although forskolin could induce ovulations and could induce significantly higher release of cAMP than LH, it resulted in a lower ovulation rate than receptor-specific LH. LH plus IBMX also induced significantly higher cAMP release than LH, as did forskolin, and resulted in a higher ovulation rate than both LH and forskolin. These findings suggest, not only that cAMP production alone is sufficient for ovulation, but also that the receptor specificity of the cAMP production is important for the number of ovulations. Since tissue levels of cAMP peak several hours before ovulation, the cAMP is probably inducing a metabolic pathway leading to ovulation.

Tissue distribution and hormonal regulation of messenger ribonucleic acid for regulatory and catalytic subunits of adenosine 3',5'-monophosphate-dependent protein kinases during ovarian follicular development and luteinization in the rat.

cDNA probes specific for the regulatory (R) subunits [RII51; (mol wt, 51,000) and RI (mol wt, 49,000)] and a catalytic (C alpha) subunit of cAMP-dependent protein kinases were used to analyze the hormonal regulation, tissue distribution, and content of mRNAs for these kinase subunits in the rat ovary. Filter hybridization assays demonstrated that mRNA specific for RII51 increased in both thecal and granulosa cells of preovulatory (PO) follicles, then declined precipitously (less than 10-fold) in both cell types within 7 h after an ovulatory (10-IU) dose of hCG and remained low in corpora lutea. Dose-response studies showed that doses of FSH greater than 2 micrograms were required to increase RII51 mRNA in granulosa cells of hypophysectomized (H) rats, whereas doses of 0.5-1.0 micrograms were effective in granulosa cells of H estradiol-treated (HE) rats. At all doses (0.5-50 micrograms) of FSH administered, RII51 mRNA was 4 times higher in granulosa cells of HE rats than in H rats. Solution hybridization assays demonstrated that the concentration of RII51 mRNA increased 6-fold from 20 molecules/granulosa cell in H rats to 120 molecules/cell in H rats treated with estradiol and FSH (HEF). Transcription assays using nuclei of granulosa cells demonstrated further that the 5- to 10-fold increases in RII51 mRNA content in estradiol-/FSH-induced granulosa cells was associated with increased transcription of the RII51 gene. In thecal cells of small antral (SA), PO, and luteinizing follicles, changes in the content of mRNA for RI and C alpha kinase subunits showed a pattern similar to that for RII51. However, in granulosa cells, mRNA specific for RI and C alpha was highest in SA follicles, declined in PO follicles, and remained unchanged during luteinization. The content of mRNA for RI (45-70 molecules/cell) and C alpha (10 molecules/cell) also changed less than 2-fold in granulosa cells of H, HE, and HEF rats. In summary, these results indicate that the content of RII51 mRNA is hormonally regulated in both thecal cells and granulosa cells during follicular development and luteinization. Low concentrations of gonadotropins increase RII51 mRNA, whereas an ovulatory dose of hCG causes mRNA for RII51 to decrease rapidly. In granulosa cells, induction of mRNA for RII51, but not that for RI and C alpha is induced by the actions of estradiol and FSH, and involves increased transcription of the RII51 gene.

Prostaglandin endoperoxide synthase in rat ovarian follicles: content, cellular distribution, and evidence for hormonal induction preceding ovulation.

These studies were undertaken to determine if the content of prostaglandin endoperoxide (PGH) synthase (PGS) was regulated in rat ovarian follicles in a time-, tissue-, and dose-dependent manner. For quantitation and localization of the enzyme by immunoblot and immunofluorescent analyses, respectively, a polyclonal antibody to highly purified (95%) ovine seminal vesicle PGS [mol wt, (Mr), 72,000] was generated in a rabbit, and an immunoglobulin G fraction of the antiserum was purified by elution from a PGS affinity resin. Soluble cell extracts were prepared from: 1) small antral (SA) and preovulatory (PO) rat follicles before and at selected time intervals after exposure to increasing doses (0.25-10.0 IU) of hCG, 2) granulosa and thecal cells of these same follicles, and 3) granulosa cells of hypophysectomized (H) rats before and after treatment with estradiol (HE), estradiol and FSH (HEF), and 10 IU hCG. Immunoblot analyses demonstrated that the content of PGS (Mr, 72,000) was low (negligible) in granulosa and thecal cells of SA and PO follicles, was induced preferentially (approximately 15-fold) in granulosa cells between 1 and 7 h after hCG treatment (0.5, 2.0, or 10 IU), and declined between 12-24 h after administration of 10 IU hCG, reaching undetectable amounts in corpora lutea isolated 48 h after hCG treatment. PGS (Mr, 72,000) was also induced by 10 IU hCG in granulosa cells of HEF rats, but was low in granulosa cells of H, HE, and HEF rats. In contrast, prostacyclin synthase was present in granulosa cells of preantral (H and HE rats), SA, and PO follicles, was not induced by hCG, and was highest in thecal cells. Immunofluorescent analyses confirmed both the tissue-specific localization and induction of PGS in granulosa cells of rat PO follicles treated with 10 IU hCG and the subcellular fractionation analyses showing that the PGS that was induced by hCG was localized primarily to a membrane (plasma membrane?) fraction of granulosa cells. Immunofluorescent data also demonstrated immunoreactive PGS in the vascular tissue of the rat corpus luteum but not in the luteal cells. Results of these studies document unequivocally that the synthesis of prostaglandins that is increased by LH/hCG in rats preceding ovulation is associated with an increased PGS content, that induction of the induced enzyme is transient, and that the enzyme is primarily localized to granulosa cell membrane fractions.

Cholesterol side-chain cleavage P450 messenger ribonucleic acid: evidence for hormonal regulation in rat ovarian follicles and constitutive expression in corpora lutea.

Using an affinity-purified antibody against cholesterol side-chain cleavage P450 (P450scc) and a human P450scc cDNA probe, 11 rat P450scc cDNA clones were identified and isolated from our rat granulosa cell lambda gt11 cDNA expression library. Two clones were plaque purified and subcloned into pBR322. One of these P450scc cDNA clones, approximately 1.2 kilobases (kb) in size, was used as a probe for Northern and filter hybridization assays to analyze the tissue distribution and hormonal regulation of P450scc mRNA in rat ovarian follicles and corpora lutea. Northern transfers revealed a single P450scc mRNA species about 2.0 kb in size. Filter hybridization assays showed that P450scc mRNA was low in granulosa cells and thecal cells of small antral follicles, was increased in both tissues of preovulatory follicles, and was rapidly (within 7 h) and maximally increased (30-fold) during hCG-induced luteinization. P450scc enzyme and mRNA were also elevated in corpora lutea isolated from pregnant rats (days 4-22 of gestation) and rats 1 day after parturition (day 23). The elevation of P450scc enzyme and mRNA was maintained despite the marked decline in serum progesterone concentrations between days 19-22, suggesting that once P450scc mRNA is induced in luteal tissue it may be constitutively expressed. Administering hormones to granulosa cells in culture and to hypophysectomized immature rats in vivo demonstrated that the induction of P450scc mRNA by FSH in granulosa cells was time, dose, and estradiol dependent. High doses of FSH acting on estradiol-primed cells gave the greatest response. The increase in P450scc mRNA in cultured granulosa cells was also stimulated by forskolin and was directly associated with increased synthesis of cAMP and progesterone accumulation. Thus, whereas the induction of P450scc mRNA in granulosa cells was dependent on hormones and cAMP, the maintenance of P450scc mRNA and P450scc protein in corpora lutea appears to involve constitutive expression of P450scc mRNA.

Isoform-specific regulation of the CCAAT/enhancer-binding protein family of transcription factors by 3',5'-cyclic adenosine monophosphate in Sertoli cells.

The C/EBP (CCAAT/enhancer-binding protein) family of transcription factors is important for differentiation, lipid biosynthesis, and metabolism. Here, we demonstrate for the first time the presence of C/EBP alpha, beta, delta, and zeta messenger RNA (mRNA) and protein in Sertoli cell primary cultures. Treatment with FSH or 8-CPTcAMP strongly induced C/EBP beta mRNA above basal levels with rapid and transient kinetics in Sertoli cell primary cultures as well as in whole testes from hypophysectomized rats. Whereas C/EBP beta mRNA was induced approximately 50-fold, C/EBP delta mRNA was induced 5- to 8-fold by cAMP in Sertoli cells. Messenger RNA for C/EBP beta and delta were induced by inhibition of protein synthesis with cycloheximide and cycloheximide acted synergistically with cAMP. Immunoblots with C/EBP antibodies demonstrated a strong induction of C/EBP beta, delta, and zeta by cAMP. Electrophoretic mobility shift analysis of nuclear proteins from cAMP-treated Sertoli cells using a C/EBP consensus oligonucleotide and antibodies revealed specific binding of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP beta antibody. Transfections of Sertoli cells with a C/EBP reporter construct showed approximately 3-fold induction of reporter gene activity by cAMP. In contrast, the reporter gene vector with a mutated form of the C/EBP binding site, was almost unresponsive to cAMP in transfections of Sertoli cells. Furthermore, C/EBP beta expression increased the activities of two promoters known to be cAMP-responsive in Sertoli cells. Thus, the early induction of C/EBP isoforms by cAMP may play a role in FSH-dependent regulation of late response genes in Sertoli cells.

Intranasal administration of human growth hormone (hGH) in combination with a membrane permeation enhancer in patients with GH deficiency: a pharmacokinetic study.

Recombinant human GH (hGH) combined with a permeation enhancer for the nasal mucosa, sodium tauro-24,25-dihydrofusidate (STDHF), was administered intranasally (in) in six patients with classical GH deficiency. Three different doses were tested (0.2, 0.4, and 0.8 IU/kg BW). The concentration of STDHF was 1% in all doses. As a comparison, all patients received a sc injection of hGH (0.1 IU/kg BW). Blood samples were obtained at frequent intervals for up to 8 h (in doses) or 24 h (sc dose) and analyzed for the plasma concentration of hGH. All three i.n. doses gave a rapid increase in hGH with peak maxima (Cmax) at 20-30 min, and a decline to baseline within 2-3 h. In contrast, the sc dose resulted in a Cmax 2-3 h after the injection, followed by a plateau phase for 2-3 h. The baseline was reached 12-14 h after administration. The uptake [area under the curve (AUC)] was considerably lower for all three in doses, i.e. 1.6-3% of the AUC obtained with the sc dose. However, the Cmax varied between 5.7 +/- 1.4% (0.8 IU/kg BW) and 15.8 +/- 2.1% (0.4 IU/kg BW) of the maximal peak with the sc dose. Of the in doses, 0.4 IU/kg BW resulted in the highest AUC and Cmax. A self-rating protocol was also used to estimate nasal sensations for up to 2 h after dosing. All sensations (itching, burning, sneezing, and running of the nose) were transient and tolerable. This study demonstrates that hGH can be administered intranasally in combination with STDHF. The in dosing results in a plasma peak of hGH very similar to the physiological endogenous peak. No side-effects were noted, other than a transient nasal irritation. Therefore, the nasal route for hGH administration offers a more physiological and more convenient alternative to injections for the treatment of GH deficiency.

Endothelial nitric oxide synthase protein is reduced in the renal medulla of two-kidney, one-clip hypertensive rats.

OBJECTIVE: We studied endothelial nitric oxide synthase (eNOS) expression in the kidneys of two-kidney, one-clip renal hypertensive rats (2K1C) before and after removal of the clip (unclipping, UC). We hypothesised that the haemodynamic changes induced by 2K1C and UC would change eNOS expression in the two kidneys. METHODS: Six weeks after inducing 2K1C, mean arterial pressure (MAP) was measured in conscious rats and hypertension reversed by UC. Left and right kidney eNOS protein in cortex and outer medulla was semi-quantified using immunoblotting. Groups were; normotensive (n = 10), 2K1C (n = 10), 3 h (n = 10), 48 h (n = 7) and 4 weeks (n = 7) after UC. The effect of 7 days of aldosterone or angiotensin II (Ang II) infusion on medullary eNOS protein was tested as well as the effect of L-NAME (nitric oxide (NO) synthase inhibitor) on medullary blood flow (MBF) in anaesthetized 2K1C. RESULTS: UC reduced MAP from 178 +/- 5 to 134 +/- 3 mmHg after 3 h and normalized MAP at 48 h and 4 weeks. The medulla from 2K1C kidneys contained about 33% less eNOS protein compared with normotensive kidneys (P < 0.05). This difference was still evident at 3 h (P < 0.05), but completely reversed at 48 h and 4 weeks after UC. Similar levels of eNOS expression were seen in the left and right kidney at all time points. Cortical eNOS was increased in kidneys from 2K1C. Neither Ang II nor aldosterone affected eNOS expression in the medulla. MBF was under similar influence of NO in 2K1C compared with normotensive kidneys. CONCLUSIONS: 2K1C is associated with reduced levels of eNOS protein in the renal medulla of both clipped and contralateral kidney. eNOS expression in right and left kidney was not changed despite expected large changes in haemodynamics of the two kidneys. The reduced level of eNOS may be associated with a reduction in MBF and thus be of patho-physiological importance in renovascular hypertension.

The expression of c-myc during follicular growth and luteal formation in the rat ovary in vivo.

The processes of folliculogenesis and formation of corpora lutea involve proliferation and differentiation of the follicular cells. The expression of several oncogenes is associated with the proliferative phase in many cell types. The present study examined the expression and hormonal regulation of the c-myc proto-oncogene during follicular development and the luteal phase of pseudopregnancy. Follicular development was initiated by pregnant mare's serum gonadotropin (PMSG) in immature rats followed two days later by the injection of human chorionic gonadotropin (hCG) to induce ovulation and luteal formation. Ovaries were collected at different time points and the content and distribution of c-myc mRNA/protein were examined. C-myc increased rapidly after the administration of both PMSG and hCG, but the effect of PMSG was less pronounced. The increase after PMSG was transient and localized primarily to the granulosa cells of developing follicles. The ovulatory dose of hCG resulted in a rapid and substantial increase of c-myc mRNA and protein with maximal levels at 1 h and 2-4 respectively. At this stage, the c-myc protein was localized to the follicular cells, the surface epithelium and, to some extent, to the interstitial tissue. There was a subsequent decrease prior to ovulation. The luteal phase was characterized by decreasing levels of c-myc with increasing luteal age. In order to examine the involvement of specific hormones in the regulation of c-myc, hypophysectomized, immature rats were injected sequentially with estradiol (E2) and follicle-stimulating hormone (FSH). Hypophysectomy resulted in a decrease of c-myc compared with intact animals. The administration of E2 resulted in an increase of c-myc mRNA and protein. The subsequent treatment with FSH did not result in a further increase and the levels remained at the same level as with E2 only. However, an ovulatory dose of hCG to E2 and FSH primed animals resulted in an additional increase of c-myc mRNA and protein. The levels after E2 and FSH were considerably lower compared with those of untreated ovaries of intact, immature animals, suggesting the involvement of other endocrine and paracrine factors. The presence of proliferating cell nuclear antigen in cell extracts indicated that the expression of c-myc was associated with phases of increased proliferation of follicular cells after hormonal stimulation. The results demonstrate that c-myc is regulated by hormones (E2, gonadotropins) in the rat ovary during follicular development to the preovulatory stage. The pronounced increase prior to ovulation also suggests a role for c-myc in the regulation of proliferative events involved in luteal formation.

Lung to blood passage of human growth hormone after intratracheal instillation: stimulation of growth in hypophysectomized rats.

The passage from the lower respiratory tract into the blood of human GH (hGH; M(r) = 22,000) and bovine serum albumin (BSA; M(r) = 67,000) was assessed after intratracheal instillation in adult rats. The plasma level of immunoreactive hGH reached its maximum at 0.5-1 h after instillation and had almost disappeared within 24 h. Higher plasma levels were obtained in male rats than in female rats resulting in a higher total lung passage of hGH in male rats than in female rats (means +/- S.D.; 6.0 +/- 1.7% vs 3.3 +/-1.2%, P<0.01). The plasma level of BSA showed a different pattern, with a maximum at 16-24 h after instillation and a total lung passage of 4.3 +/- 1.7% of the given dose for both sexes. The plasma levels of hGH increased nonlinearly with increasing dose instilled in the dose range 36-720 micrograms/kg body weight. When hGH was instilled daily at a dose of 720 micrograms/kg body weight to hypophysectomized rats for 1 week, they responded with a significant increase in body weight when compared with hypophysectomized control rats (16.8 +/- 4.2 g vs -1.8 +/- 2.4 g, P<0.001). The results demonstrate that, despite their different molecular weights, hGH and BSA pass through the lower respiratory tract into the circulation with similar efficiencies in the rat. However, the lung passage of hGH, unlike that of BSA, showed sexual dependency, an earlier plasma concentration maximum and a tendency of the passage to saturation with increasing dose instilled.(ABSTRACT TRUNCATED AT 250 WORDS)

Forskolin effects on the cAMP system and steroidogenesis in the immature rat ovary.

The diterpene forskolin was found to activate the adenylate cyclase system in intact tissue and membrane preparations of the immature rat ovary. The cyclic AMP (cAMP) response reached a maximal level after 5 min and no decline was observed even after 4 h of incubation. Forskolin stimulated production of both progesterone and testosterone in a pattern similar to that produced by luteinizing hormone (LH) or dibutyryl-cAMP (dbcAMP). In combination with LH, follicle-stimulating hormone (FSH) or prostaglandin E2 (PGE2), forskolin potentiated the hormone effects on adenylate cyclase activity in membrane preparations. Pretreatment with LH or PGE2 desensitized the cells to further hormone stimulation, while the forskolin response was unaffected. Pre-exposure to forskolin did not desensitize the cells to a subsequent stimulation by LH or PGE2. The presence of 8-bromo-cAMP (brcAMP) in the preincubation medium reduced the subsequent hormone response. These results demonstrate a rapid and sustained activation of the adenylate cyclase system by forskolin in the rat ovary. The steroidogenic response was similar to that of known stimulators of ovarian cells (LH, dbcAMP). The inability of forskolin to induce desensitization of the adenylate cyclase system demonstrates, however, important differences between hormone and non-hormone activation. Consequently, forskolin can be a useful tool for investigation of the mechanisms involved in the desensitization process.

Involvement of cyclic AMP and protein synthesis in the desensitization of the prepubertal rat ovary.

Luteinizing hormone (LH) stimulates cyclic AMP (cAMP) production in ovarian cells. After the initial stimulation there is a development of refractoriness (desensitization) to a subsequent LH stimulation. The present investigation was designed to study the role of cAMP in this desensitization process and the influence of protein-synthesis inhibitors. Ovaries from 23-day-old rats were preincubated in buffer, dibutyryl-cAMP, 8-bromo-cAMP or LH. After a washing period in buffer, the ovaries were incubated in the presence of LH. Those ovaries preexposed to the cAMP analogues showed a reduced cAMP-production to LH. This reduction was found to be both dose- and time-dependent, but was never as marked as after preincubation with LH. Desensitization developed more slowly after preincubation with cAMP than with LH. Inhibitors of protein synthesis (puromycin, cycloheximide) completely abolished the cAMP-induced desensitization but only partially prevented the LH-induced desensitization. These results show that processes, dependent and independent of cAMP, are involved in the desensitization after LH stimulation of the prepubertal rat ovary.