Expression of proteoglycans in two types of ameloblastoma: novel Immunohistochemical findings.

Alterations in cellular and extracellular matrix components play an important role during tumorigenesis; proteoglycans are included among these components. Ameloblastomas are odontogenic tumors distinguished as invasive and infiltrative neoplasms and are divided into different histological types, the most common of which are the unicystic ameloblastoma and the conventional ameloblastoma. The aim of this study was to identify the presence of two proteoglycans, perlecan and biglycan, in different types of ameloblastoma. Using immunohistochemistry, we determined the presence of both proteins in 28 unicystic ameloblastomas and 23 conventional ameloblastomas. We identified the cytoplasmic and nuclear presence of perlecan and the cytoplasmic presence of biglycan in both types of ameloblastoma. The mean values of immunoexpression were higher in the conventional type compared to the unicystic type. Neither the presence of biglycan in ameloblastomas nor the nuclear presence of perlecan in any odontogenic tumor has previously been reported. The differential immunoexpression of perlecan and biglycan in these types of ameloblastomas suggests their participation in the developmental process of these tumors.

Effects of systemic administration or intrabursal injection of serotonin on puberty, first ovulation and follicular development in rats.

To elucidate the role of serotonin in the onset of puberty, the effects of both systemic and in-ovarian bursa administration of serotonin on the neuroendocrine mechanism that modulates the onset of puberty, follicular development and first ovulation were evaluated. Two experiments were carried out. For the first, 25 or 37.5 mg kg⁻¹ of bodyweight of serotonin creatinine sulfate was administered by a subcutaneous route to 30-day-old female rats. In the second experiment, serotonin creatinine sulfate was administered directly into the ovarian bursa of 34-day-old female rats. Systemic administration of 25 or 37.5 mg kg⁻¹ of serotonin creatinine sulfate induced a delay in the ages of vaginal opening and first vaginal oestrus, a decrease in the number of ovulating animals, and serum concentrations of FSH, LH, oestradiol and progesterone. An increase in the number of Class 3 (>500 µm) and atretic follicles was observed in the ovaries of these animals. The administration of serotonin creatinine sulfate in the ovarian bursa did not modify the onset of puberty and ovulation, but a reduced serum concentration of oestradiol was observed. Our results suggest that serotonin acts on the components of the hypothalamus-hypophysis-ovary axis by modulating follicular development, ovarian functions and the onset of puberty.

Syndecan-1 (CD138) and Ki-67 expression in different subtypes of ameloblastomas.

Ameloblastoma is the most frequent odontogenic tumor and is considered a benign, but locally invasive, neoplasm with variable clinico-pathological expression. Syndecan-1 is a cell surface proteoglycan that binds cells to the extracellular matrix and its expression is down-regulated in many cellular transformation models. The aims of this study were to examine the pattern of syndecan-1 expression, to evaluate the proliferating activity in a large series of solid/multicystic (SA) and unicystic ameloblastomas (UA), and to study its possible correlation to their biological behavior. Immunohistochemical studies were performed for syndecan-1 (clone MI15) and Ki-67 (clone MIB-1) in 120 ameloblastomas (75 SA and 45 UA). The salient finding was that expression of syndecan-1 was related to the histological subtype of tumors, as there was a lower expression in SA (40.2%) as compared to UA (49.7%) (p<0.05). These findings did not correlate with Ki-67 expression, as this was similar in both types of ameloblastomas. Our results suggest that the reduced expression of syndecan-1 supports the view that SA has a more aggressive biological behavior than the UA. The lack of correlation between reduction of the syndecan-1 and Ki-67 index may be due to the different histomorphologies of both types of ameloblastoma, and more studies are necessary to better understand the role of this protein in the biological behavior of these tumors.

Human embryonic kidney cells: stable transformation with an origin-defective simian virus 40 DNA and use as hosts for human papovavirus replication.

An origin-defective mutant DNA of simian virus 40 immortalized human embryonic kidney cells, maintaining a T protein which could function for human papovavirus BK DNA replication but not for human papovavirus JC DNA replication. Neither BK virions nor capsid proteins were produced in these cells. This may indicate that the simian virus 40 T protein in human embryonic kidney cells is competent for maintaining transformation and initiating and completing DNA replication for BK but is not competent for switching to late gene functions. Furthermore, it appears that the JC DNA replication origin cannot efficiently use the simian virus 40 T protein for its DNA synthesis, as suggested by its DNA sequence data (R. Frisque, J. Virol. 46:170-176, 1983; T. Miyamura, H. Jikoya, E. Soeda, and K. Yoshiike, J. Virol. 45:73-79, 1983).

Increasing roughness of the human breast cancer cell membrane through incorporation of gold nanoparticles.

Gold nanoparticles (AuNPs) have been proposed for use in the treatment of different types of cancer, including breast cancer. At present, neither the mechanisms of AuNP interaction with the plasma membrane surface and their delivery and intracellular distribution in cancer cells nor their effect on the plasma membrane so as to allow cell incorporation of larger amounts of AuNPs is known. The objective of this work was to study the interaction of bare 20 nm diameter AuNPs with the plasma membrane of human MCF-7 breast cancer cells, as well as their uptake, intracellular distribution, and induction of changes on the cell surface roughness. The dynamics of intracellular incorporation and the distribution of AuNPs were observed by confocal laser scanning microscopy. Changes in roughness were monitored in synchronized MCF-7 cells by atomic force microscopy high-resolution imaging at 6 hour intervals for 24 hours during a single cell cycle. The results show that bare AuNPs are capable of emitting fluorescence at 626 nm, without the need for a fluorescent biomarker, which allows monitoring their uptake and intracellular distribution until they reach the nucleus. These results are correlated with changes in cell roughness, which significantly increases at 12 hours of incubation with AuNPs, when compared with control cells. The obtained data provide bases to understand molecular processes of the use of AuNPs in the treatment of different diseases, mainly breast cancer.

Nicotinamide sensitizes human breast cancer cells to the cytotoxic effects of radiation and cisplatin.

Poly(ADP-ribose) polymerase (PARP) inhibitors enhance the effect of DNA alkylating agents on BRCA1­ and BRCA2-deficient cell lines. The aim of this study was to analyze the effect of the PARP inhibitor nicotinamide (NAM) on breast cancer cells with different BRCA1 expression or function, such as BRCA1­deficient MDA-MB-436 cells, low expression BRCA1 MCF-7 cells, and the BRCA1 wild­type MDA-MB-231 cells, to demonstrate its effects as a chemo­ or radiosensitizing agent. PARP activity was analyzed in MDA-MB-436, MCF-7 and MDA-MB-231 breast cancer cells subjected or not to NAM. Inhibition of PARP by NAM in the presence of DNA damage was examined by Alexa Fluor 488 immunofluorescence. Crystal violet assays were used to test growth inhibition and the chemo­ and radiosensitization effects of NAM were investigated using clonogenic assays. Significant differences among data sets were determined using two-tailed ANOVA and Bonferroni tests. We demonstrated that NAM reduces PARP activity in vitro, and in cells subjected or not to DNA damage, it also reduces the viability of breast cancer cell lines and synergyzes the cytotoxicity of cisplatin in MDA-MB-436 and MCF-7 cells. Downregulation of PARP1 with siRNA led to modest growth inhibition, which was further increased by cisplatin. Nicotinamide also induced radiosensitization in MDA-MB-436 and MDA-MB-231 cells. In conclusion, NAM may be used as a chemo­ or radiosensitizing agent regardless of the BRCA1 status in breast cancer.

An alternative sigma factor controls transcription of flagellar class-III operons in Escherichia coli: gene sequence, overproduction, purification and characterization.

Based on the studies of the FliA protein in Bacillus subtilis (Bs) and Salmonella typhimurium (St), the Escherichia coli (Ec) fliA gene has been proposed to encode a flagellar-specific sigma factor, sigma 28. In this study, the complete nucleotide (nt) sequence of Ec fliA was determined. The fliA coding region consists of 717 nt starting with a GTG start codon and ending with a TAA stop codon. The gene product is predicted to be 239 amino acids (26,435 Da). Sequence comparison between Ec FliA and the sigma 28 of St revealed 93.7% identity. Gene fliA was amplified by the polymerase chain reaction, subcloned into expression vector pT7-7, and overexpressed. The overproduced 28-kDa FliA protein, recognized by the St anti-sigma 28 antibody, was purified to homogeneity. The purified protein was able to initiate transcription from the tar promoter in the presence of RNP core enzyme. We conclude that FliA functions as an alternative sigma factor sigma 28 which is specific for flagellar operons in Ec.

Gene sequence, overproduction, purification and determination of the wild-type level of the Escherichia coli flagellar switch protein FliG.

The flagellar motor switch in Escherichia coli and Salmonella typhimurium controls swimming behavior by regulating the direction of flagellar rotation. The switch is a complex apparatus composed of at least three proteins--FliG, FliM and FliN. During chemotactic behavior, the switch responds to signals transduced by the chemotaxis sensory signaling system. CheY, the chemotaxis response regulator, is thought to act directly on the switch to induce tumbles in the swimming pattern, but physical interaction of CheY and switch proteins has not been shown. We have undertaken this work to develop the molecular tools to investigate CheY binding to switch proteins, as well as to understand more about the structure and function of the switch. We present here the sequences of the fliG gene and its protein product, the engineering and amplification of fliG by the polymerase chain reaction (PCR) and its subcloning, and the overproduction, purification and determination of the wild-type (wt) level of the FliG protein. The sequence data revealed a 91.8% amino acid (aa) identity between E. coli and S. typhimurium FliG. Engineering and amplifying fliG by PCR allowed convenient cloning into an efficient expression vector. FliG was successfully overproduced and purified to > 98% purity. Polyclonal antibodies (Ab) were generated against purified FliG and used in quantitative Western blots to determine that the wt expression level of fliG results in about 3700 FliG copies per cell. Purified FliG and anti-FliG Ab will be useful for direct biochemical analyses of CheY-switch protein interaction.

Oestrogens regulate pituitary alpha2,3-sialyltransferase messenger ribonucleic acid levels in the female rat.

Follicle-stimulating hormone (FSH) is synthesized by the anterior pituitary gland in multiple molecular forms. Increased acidic/sialylated FSH charge isoforms are associated with conditions characterized by a low oestrogen output. In the present study, we analysed the dynamics of the changes in mRNA levels of the enzyme Galbeta1,3[4]GlcNAc alpha2,3-sialyltransferase (2,3-STase) (one of the enzymes that incorporate sialic acid residues into the FSH molecule) in intact and ovariectomized rats. The anterior pituitaries of 4-day regularly cyclic adult female Wistar rats were obtained at 1000 h on the days of pro-oestrus (P), oestrus (O), dioestrus 1 (D1) and dioestrus 2 (D2), at 0200 h, 1400 h, 1800 h and 2200 h on D1, at 1800 h on day of O and at 1000 h after 7, 14, 21, 28 and 45 days of oophorectomy performed on the morning of P. Total RNA was isolated from each gland and the 2,3-STase levels were measured by Northern blot hybridization analysis employing a 346-base pair cDNA probe encoding for a non-conserved amino acid sequence of the catalytic domain of the enzyme. Maximal levels of the enzyme mRNA were detected at 1000 h on D1; thereafter, they progressively decreased by 60% during the ensuing 24 h, reaching the lowest concentration values (26% of the maximally observed level on D1) at 1000 h on day of P and remaining unchanged during the morning of O. Administration of the potent oestradiol receptor antagonist ICI 182,780 at 1000 h on D1 completely reverted the time-dependent decrease in 2,3-STase mRNA levels observed during the afternoon of D1, whereas oestradiol benzoate administered at 1000 h on day of O significantly reduced the enzyme mRNA levels (to 21% of the levels detected in vehicle-treated controls). In ovariectomized rats, the alpha2,3-STase mRNA progressively increased from day 21 to day 45 post castration. Administration of oestradiol benzoate on day 28 after oophorectomy significantly reduced the 2,3-STase mRNA levels (to 36% of the levels detected in vehicle-injected controls); ICI 182,780 partially counteracted this oestradiol-mediated effect. The dynamics of these changes in 2,3-STase mRNA levels partially correlated with changes in the relative abundance of the FSH charge isoforms separated by preparative chromatofocusing of anterior pituitary extracts, particularly in glands obtained during the morning of P and O. These data demonstrate for the first time that pituitary 2,3-STase is a hormonally-regulated enzyme and that the changes in transcription and/or stability of its mRNA may be involved, in part, in the post-translational processing of the FSH molecule during certain physiological conditions.

Effects of gonadotrophin-releasing hormone, recombinant human activin-A and sex steroid hormones upon the follicle-stimulating isohormones secreted by rat anterior pituitary cells in culture.

FSH is produced and secreted from the anterior pituitary gland of rats in multiple molecular forms. At times of high gonadotrophin-releasing hormone (GnRH) and oestrogen output (e.g. the morning of the day of pro-oestrus) the pituitary increases the production of FSH isoforms with isoelectric point (pI) values greater than 5.0, whilst sex steroid deprivation leads to the production of strongly acidic and less in-vitro biologically active FSH molecules. It is not known, however, whether sex steroids modulate the production of specific FSH isoforms by a direct action at the pituitary level or indirectly through altering the rate of synthesis and/or secretion of GnRH. In order to obtain some insight on this issue, we examined the charge heterogeneity of FSH secreted by cultured pituitary cells exposed to different FSH-releasing factors, oestradiol-17 beta and progesterone, alone or in different time-sequenced combinations. Anterior pituitary glands from 21-day-old female rats were enzymatically dispersed into a single cell suspension and cultured for 5 days. During days 1 to 3, cells were incubated in the absence of factors or steroid hormones; on days 3 to 4, cells were incubated in the absence (controls) or presence of either oestradiol-17 beta (3.67 nmol/l) or oestradiol-17 beta plus progesterone (3.67 and 31.8 nmol/l respectively). Finally, during days 4 to 6, GnRH (10 nmol/l) or recombinant human activin-A (2 nmol/l) were added to half of all culture wells. Media from each cell group were concentrated and the several forms of secreted FSH were then separated by polyacrylamide gel isoelectric focusing (pH range 6.5-4.0) and quantitated. All media concentrates contained several forms of immunoactive secreted FSH focusing within a pH range of 6.44-4.23. A large amount (51-76%) of total FSH recovered focused within a pI range of 4.9-4.0 (area 3), whilst 20-43% and 4-8% of the total were identified within pI range of 5.9-5.0 (area 2) and 6.5-6.0 (area 1) respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of neonatal androgenization on the chromatofocusing pattern of anterior pituitary FSH in the female rat.

Anterior pituitary glands were removed from neonatally androgenized (100 micrograms testosterone propionate) female rats and normal controls at 5, 10, 18, 21, 30, 60 and 90 days of age, and the multiple forms of FSH present within them were separated by chromatofocusing (pH range 7.5-4.0). Additional pituitary glands from intact adult males (90 days old) were also studied for comparative purposes. All animal groups exhibited multiple forms of immunoactive FSH within a pH range of 7.5-4.0, as well as an additional FSH form obtained after the addition of 1.0 mol NaCl/l to the chromatofocusing column (salt peak). In animals 5-30 days old (controls and androgenized) the majority of FSH applied to the chromatofocusing columns was recovered within the salt peak (45-85% of total FSH immunoactivity recovered). However, as the animals aged, more FSH immunoactivity focused within less acidic regions (isoelectric point (pI) 5.9-5.0); pituitaries from animals 60 days old contained the greatest proportion of FSH focused within this pH range (controls, 39.2 +/- 0.6%; androgenized, 23.1 +/- 0.9% of total immunoactivity recovered; P less than 0.03 vs animals 30 days old for both experimental groups). This shift towards less acidic FSH was attenuated in androgenized animals compared with the controls (P less than 0.01). In control adult rats, the chromatofocusing distribution pattern of pituitary FSH varied according to the day of the oestrous cycle. Pituitary extracts from control rats decapitated during the morning of pro-oestrus, oestrus and day 1 of dioestrus exhibited the highest proportion of immunoactive FSH (23.2-28.8% of total) focused within a pH range of 5.9-5.0, whilst only 10.4-11.6% of FSH from androgenized rats and those on day 1 of dioestrus was recovered within this pH range (P less than 0.05). In control animals decapitated during the morning of pro-oestrus and oestrus, 10-26% of FSH focused within the most alkaline region (pI 7.5-6.0); the chromatofocusing pattern of pituitary FSH from the neonatally androgenized animals was characteristic, in that no more than one peak (1.5 +/- 0.5% of total) was detected in this alkaline region. In the adult male rats, the majority of pituitary FSH eluted from the chromatofocusing columns within a pH of 4.9-4.0 (52.4 +/- 1.2% of total FSH immunoactivity) and the salt peak (pH less than 4.0) (33.1 +/- 2.4 of total). All FSH isoforms obtained after chromatofocusing represented alpha and beta dimers as disclosed by size exclusion chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)

The oestrogenic effects of gestodene, a potent contraceptive progestin, are mediated by its A-ring reduced metabolites.

Gestodene (17 alpha-ethynyl-13 beta-ethyl-17 beta-hydroxy-4, 15-gonadien-3-one) is the most potent synthetic progestin currently available and it is widely used as a fertility regulating agent in a number of contraceptive formulations because of its high effectiveness, safety and acceptability. The observation that contraceptive synthetic progestins exert hormone-like effects other than their progestational activities, prompted us to investigate whether gestodene (GSD) administration may induce oestrogenic effects, even though the GSD molecule does not interact with intracellular oestrogen receptors (ER). To assess whether GSD may exert oestrogenic effects through some of its neutral metabolites, a series of experimental studies were undertaken using GSD and three of its A-ring reduced metabolites. Receptor binding studies by displacement analysis confirmed that indeed GSD does not bind to the ER, whereas its 3 beta,5 alpha-tetrahydro reduced derivative (3 beta GSD) interacts with a relative high affinity with the ER. The 3 alpha,5 alpha GSD isomer (3 alpha GSD) also binds to the ER, though to a lesser extent. The ability of the A-ring reduced GSD derivatives to induce oestrogenic actions was evaluated by the use of two different molecular bioassays: (a) transactivation of a yeast system co-transfected with the human ER alpha (hER alpha) gene and oestrogen responsive elements fused to the beta-galactosidase reporter vector and (b) transactivation of the hER alpha-mediated transcription of the chloramphenicol acetyl transferase (CAT) reporter gene in a HeLa cells expression system. The oestrogenic potency of 3 beta GSD was also assessed by its capability to induce oestrogen-dependent progestin receptors (PR) in the anterior pituitary of castrated female rats. The results demonstrated that 3 beta GSD and 3 alpha GSD were able to activate, in a dose-dependent manner, the hER alpha-mediated transcription of both the beta-galactosidase and the CAT reporter genes in the yeast and HeLa cells expression systems respectively. In both assays the 3 beta derivative of GSD exhibited a significantly greater oestrogenic effect than its 3 alpha isomer, while unchanged GSD and 5 alpha GSD were completely ineffective. Neither 3 beta GSD nor 3 alpha GSD exhibited oestrogen synergistic actions. Interestingly, the pure steroidal anti-oestrogen ICI-182,780 diminished the transactivation induced by 3 beta GSD and 3 alpha GSD in the yeast expression system. Furthermore, administration of 3 beta GSD resulted in a significant increase of oestrogen-dependent PR in the anterior pituitaries of castrated rats in comparison with vehicle-treated animals. The characteristics of the 3 beta GSD-induced PR were identical to those induced by oestradio benzoate. The overall results demonstrate that 3 beta GSD and its 3 alpha isomeric alcohol specifically bind to the ER and possess a weak intrinsic oestrogenic activity, whereas unmodified GSD does not. The data contribute to a better understanding of the GSD mechanism of action and allow the hypothesis to be advanced that the slight oestrogenlike effects attributable to GSD are mediated by its non-phenolic, tetrahydro reduced metabolites.

Human estrogen receptor regulation in a yeast model system and studies on receptor agonists and antagonists.

An expression system that utilized yeast copper metallothionein promoter and ubiquitin fusion technology to express the human estrogen receptor gene in yeast is described. We have studied the biochemical and transcriptional regulatory properties of the human estrogen receptor. The biochemical properties of the yeast expressed receptors are identical to the receptors isolated from human tissue. Estradiol mediated activation of transcription by the receptor was studied by a reporter beta-galactosidase gene where expression was under the control of estrogen response elements. Using this expression system and a hyperpermeable yeast strain we have studied the effects of various antiestrogens on the regulation of estrogen receptor function. We demonstrate that tamoxifen and ICI 164,384 are capable of binding to the receptor but neither antiestrogen was able to block the estradiol mediated increase in transcription. In fact, both antiestrogens exerted weak agonist activity in this system.

FlhD/FlhC-regulated promoters analyzed by gene array and lacZ gene fusions.

The Escherichia coli transcriptional regulatory complex FlhD/FlhC, initially identified as a flagella-specific activator, is a global regulator involved in many cellular processes. Using gene arrays, lacZ gene fusions and enzyme assays, eight new targets of FlhD/FlhC were recognized. These are the transporter for galactose (MglBAC), the rod-shape determination proteins (MreBCD), malate dehydrogenase, and several enzymes involved in anaerobic respiration (glycerol 3-phosphate dehydrogenase, GlpABC; periplasmic nitrate reductase, NapFAGHBC; nitrite reductase, NrfABCDEFG; dimethyl sulfoxide reductase, DmsABC; and the modulator for hydrogenases, HydNHypF).

In vitro metabolism of gestodene in target organs: formation of A-ring reduced derivatives with oestrogenic activity.

Gestodene (13beta-ethyl-17alpha-ethynyl-17beta-hydroxy-4,5-gonadien-3-one), the most potent progestin ever synthesized, stimulates breast cancer cell growth through an oestrogen receptor-mediated mechanism, and its use in hormonal contraception has been associated with side effects attributable to oestrogenic actions. These observations have remained controversial, since gestodene does not bind to the oestrogen receptor or exert oestrogen-like activities. Recently, we have demonstrated that non-phenolic gestodene derivatives interact with oestrogen receptors and induce oestrogenic effects in cell expression systems. To assess whether gestodene is biotransformed to metabolites with intrinsic oestrogenic potency, [3H]- and [14C]-labelled gestodene were incubated in vitro with rat anterior pituitary, hypothalamus and ventral prostate homogenates under different experimental conditions. The most remarkable finding was the isolation and identification of 3beta,5alpha-tetrahydrogestodene and 3alpha,5alpha-tetrahydrogestodene as metabolic conversion products of gestodene, presumably with 5alpha-dihydrogestodene as intermediate. The overall results seem to indicate that the weak oestrogenic effects attributable to gestodene could be mediated by its tetrahydro metabolites.

Assessment of the oestrogenic activity of the contraceptive progestin levonorgestrel and its non-phenolic metabolites.

Levonorgestrel (13beta-ethyl-17alpha-ethynyl-17beta-hydroxy-4-gonen-3-one), a potent contraceptive progestin stimulates growth and proliferation of cultured breast cancer cells through a receptor-mediated mechanism, even though levonorgestrel does not bind to the oestrogen receptor (ER). To assess whether the oestrogen-like effects induced by this synthetic progestin are exerted via its metabolic conversion products, we studied the binding affinity of three A-ring levonorgestrel derivatives to the ER and their capability to transactivate an oestrogen-dependent yeast system co-transfected with the human ER gene and oestrogen responsive elements fused to a beta-galactosidase reporter vector. The results demonstrated that the 3beta,5alpha reduced levonorgestrel derivative and to a lesser extent its 3alpha isomer interact with the oestrogen receptor, with a significantly lower relative binding affinity (2.4% and 0.4%, respectively) than that of oestradiol (100%), while levonorgestrel does not. Both levonorgestrel metabolites were able to activate, in a dose-dependent manner, the beta-galactosidase reporter gene in the yeast expression system, an effect that was precluded by a steroidal antioestrogen. The oestrogenic potency of levonorgestrel metabolites was significantly lower (750-fold) than that of oestradiol. Furthermore, high doses of 3beta,5alpha levonorgestrel (2.5 mg/day/6 days) induced an increase of oestrogen-dependent progestin receptor in the anterior pituitary of castrated rats. The overall data offer a plausible explanation for the weak oestrogenic effects induced by high, non-pharmacological doses of levonorgestrel.

Changes in monoaminergic activity in the anterior, medium and posterior hypothalamus, gonadotropins levels and ovarian hormones during puberty of the female rat.

The aim of present study is the analysis of monoamines concentrations changes in the anterior, medium and posterior hypothalamus, as well as changes in serum gonadotropins levels, ovarian steroids and follicular growth during the prepubertal development of the female rat. Noradrenergic activity in the anterior, medium and posterior hypothalamus reached highest level at day 13 after birth, followed by a subsequent decrease from day 15 to 19 and an increase on days 22 and 27 postnatal. At day 1, neural activity in the medium hypothalamus was higher than the activity in the anterior and posterior hypothalamus. Serotoninergic activity in three portions of the hypothalamus was higher throughout the prepubertal development. Follicle-stimulating hormone and luteinizing hormone serum levels increased between days 11 and 17 and decreased from day 19 to 36. The concentration of 17beta-estradiol was consistently low throughout the prepubertal development and increased at day 39 after birth. These results indicate that during the prepubertal development of the rat, the three regions of the hypothalamus show significant changes in the monoaminergic neural activity. There is an inverse relationship between the noradrenergic activity on the anterior and medium hypothalamus and serotoninergic activity in the posterior hypothalamus with ovarian steroids during sexual maturation. These changes may be linked to the development of the neuroendocrine processes that modulate gonadotropin secretion and ovarian function.

Endocrine regulation of gonadotropin glycosylation.

The pituitary gonadotropins--luteinizing hormone and follicle-stimulating hormone--as well as the placental choriogonadotropin belong to the family of glycoprotein hormones. These structurally related hormones, which regulate several major reproductive functions of the body, are heterodimers consisting of a common alpha-subunit noncovalently bound to a beta-subunit. The N- and O-linked oligosaccharide chains of these gonadotropins play an important role in intracellular folding, assembly, secretion, metabolic clearance, and biological activity of the hormone. Gonadotropin glycosylation is a highly complex process; within the gonadotropes it is modulated by a variety of extrapituitary factors of hypothalamic and gonadal origin. In particular, estrogens and androgens appear to regulate terminal sialylation and/or sulfation of the oligosaccharide attachments and hence some functional properties of the gonadotropin molecule determined by these residues, i.e., metabolic clearance and in vivo biopotency. Through these extrapituitary inputs, the anterior pituitary may not only regulate the quantity but also the quality of the gonadotropin signal delivered to the gonads in a given physiologic or pathologic condition.

Sequential nitrification-denitrification process for nitrogenous, sulfurous and phenolic compounds removal in the same bioreactor.

The kinetic and metabolic behavior of an aerobic granular sludge to nitrify, denitrify and nitrify-denitrify was evaluated in batch cultures. In nitrification control, ammonium, 4-methylphenol and sulfide were consumed efficiently (∼100%) and recovered as NO3(-), CO2, S(0) and SO4(2-), respectively. In denitrification control, S(0) and nitrate were efficiently consumed and recovered as SO4(2-) and N2, respectively. Sequential nitrification-denitrification process was evaluated by applying oxic/anoxic conditions. Ammonium, 4-methylphenol and sulfide were oxidized to nitrate, CO2 and mainly S(0), respectively, under aerobic conditions. After that, anoxic conditions were established where S(0) reduced all nitrate to N2, with molecular nitrogen yield (YN2) of 1.03 ± 0.06 mg/mg NH4(+)-N consumed. This is the first study to show the capability of an aerobic granular sludge in simultaneous removal of ammonium, 4-methylphenol and sulfide by sequential nitrification-denitrification process in the same bioreactor.