Advanced phosphorus removal via coagulation, flocculation and microsieve filtration in tertiary treatment.

The applicability of microsieve technology together with coagulation and flocculation for advanced phosphorus removal was investigated. A pilot unit including a microsieve with 10 µm mesh size was operated continuously with secondary effluent from Ruhleben wastewater treatment plant in Berlin. By applying a pretreatment of 0.07-0.09 mmol/L (as metal) coagulant and 1.5-2 mg/L cationic polymer, total phosphorus values below 80 µg/L were achieved. Coagulation with polyaluminum chloride (PACl) produced a better effluent quality compared to FeCl3, as less suspended solids and less residual coagulant were found in the microsieve effluent. In addition, the transmittance of UV radiation through the water was improved by using PACl. The produced amount of backwash water was always below 3% (on average 1.6%). Under optimized mixing conditions, polymer doses of 0.6 mg/L were possible without losses in water quality and filtration performance. Microsieving with chemical pretreatment is a viable option for high quality effluent polishing.

Automatic control of the effluent turbidity from a chemically enhanced primary treatment with microsieving.

For chemically enhanced primary treatment (CEPT) with microsieving, a feedback proportional integral controller combined with a feedforward compensator was used in large pilot scale to control effluent water turbidity to desired set points. The effluent water turbidity from the microsieve was maintained at various set points in the range 12-80 NTU basically independent for a number of studied variations in influent flow rate and influent wastewater compositions. Effluent turbidity was highly correlated with effluent chemical oxygen demand (COD). Thus, for CEPT based on microsieving, controlling the removal of COD was possible. Thereby incoming carbon can be optimally distributed between biological nitrogen removal and anaerobic digestion for biogas production. The presented method is based on common automation and control strategies; therefore fine tuning and optimization for specific requirements are simplified compared to model-based dosing control.

Microsieving in primary treatment: effect of chemical dosing.

Primary and chemically enhanced primary wastewater treatment with microsieving (disc or drum filtration) was studied at the large pilot scale at seven municipal wastewater treatment plants in Europe. Without chemical dosing, the reduction of suspended solids (SS) was (on average) 50% (20-65%). By introducing chemically enhanced primary treatment and dosing with cationic polymer only, SS removal could be controlled and increased to >80%. A maximum SS removal of >90% was achieved with a chemical dosing of >0.007 mg polymer/mg influent SS and 20 mg Al(3+)/L or 30 mg Fe(3+)/L. When comparing sieve pore sizes of 30-40 µm with 100 µm, the effluent SS was comparable, indicating that the larger sieve pore size could be used due to the higher loading capacity for the solids. Phosphorus removal was adjusted with the coagulant dose, and a removal of 95-97% was achieved. Moreover, microsieving offers favourable conditions for automated dosing control due to the low retention time in the filter.

Expression and regulation of gonadotropin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acids in human granulosa-luteal cells.

The present study investigated the expression and regulation of GnRH and GnRH receptor (GnRHR) messenger RNAs (mRNAs) in human granulosa-luteal cells using reverse transcription-polymerase chain reaction (RT-PCR). Granulosa-luteal cells were aspirated from preovulatory follicles obtained from women undergoing in vitro fertilization. Two sets of primers derived from human hypothalamic GnRHR complementary DNA (cDNA) were used to amplify cDNAs from granulosa-luteal cells. PCR products corresponding to the expected sizes of GnRH were obtained from granulosa-luteal cells as well as the brain, but not from skeletal muscle cDNA. The authenticity of the PCR products was confirmed by Southern blot hybridization with internal oligonucleotide probes and by subsequent cloning and sequencing. Similarly, using four sets of primers specific for the human pituitary GnRHR cDNA, PCR products with the expected sizes were detected from both brain and granulosa-luteal cells, but not from skeletal muscle. PCR products were subsequently confirmed by Southern blot hybridization using an internal oligonucleotide probe or a cDNA probe which was obtained from screening a human pituitary cDNA library. Cloning and sequencing of the PCR product in the 3'-untranslated region revealed identical sequence with the reported human pituitary GnRHR cDNA sequence. RNA samples obtained from cells immediately after dissociation or after 2, 5, and 8 days of culture were analyzed by RT-PCR, and in all cases, both GnRH and GnRHR mRNA were detected. To investigate how gene expression of GnRH and GnRHR is regulated, we examined the effect of GnRH and hCG on GnRH and GnRHR mRNA levels in cultured human granulosa-luteal cells. Treatment with different concentrations of GnRH induced biphasic responses. Both GnRH and GnRHR mRNA were significantly increased by 1 nM, but slightly decreased by 1 microM GnRH; 1 nM GnRH also significantly inhibited progesterone production, whereas higher doses had no effect. Treatment with hCG (1 IU/ml) decreased GnRHR mRNA levels without altering the expression of the GnRH gene. These results demonstrate for the first time that 1) both GnRH and GnRHR mRNAs are expressed in human granulosa-luteal cells; 2) GnRH mRNA levels are autoregulated by GnRH; and 3) GnRHR gene expression is up-regulated by GnRH, but down-regulated by hCG. These findings provide strong evidence that GnRH is an autocrine regulator in the human ovary.

The third intracellular loop of the rat gonadotropin-releasing hormone receptor couples the receptor to Gs- and G(q/11)-mediated signal transduction pathways: evidence from loop fragment transfection in GGH3 cells.

The GnRH receptor (GnRH-R) belongs to the rhodopsin/beta-adrenergic family of G protein-coupled receptors. The intracellular domains of these receptors, particularly the regions closest to the plasma membrane in intracellular loops 2 (2i) and 3 (3i) as well as some regions located in the membrane-proximal end of the COOH-terminus, are frequently important sites for G protein coupling and specificity determination. Although studies in mouse and human GnRH-R have identified loop 2i as a critical determinant for coupling the receptor to the G(q/11)-mediated signal transduction pathway, given the functional similarity among the members of this particular G protein-coupled receptor subfamily and the fact that the GnRH-R lacks the typical intracellular COOH-terminal domain of its superfamily (a potential site for G protein coupling), we investigated the possibility that loop 3i of this receptor also participates in GnRH-R coupling to G proteins. GGH(3)1' cells, a pituitary-derived cell line that expresses a functional rat GnRH-R coupled to both Gs and G(q/11) proteins, were transiently transfected with a plasmid DNA containing a complementary DNA (cDNA) coding for the entire loop 3i of the GnRH-R as well as with other expression plasmids containing cDNAs encoding loop 3i of other Gs-, G(i/o)-, or G(q/11)-coupled receptors. The effects of coexpression of these loops with the wild-type GnRH-R on inositol phosphate (IP) production, cAMP accumulation, and PRL release were then examined. Transfection of GGH(3)1' cells with the cDNA for loop 3i of the rat GnRH-R (efficiency, 35-45%) maximally inhibited buserelin-stimulated IP turnover by 20% as well as cAMP accumulation and PRL secretion by 30%. This attenuation in cellular responses to a GnRH agonist was statistically significant (P < 0.05) compared with the responses exhibited by GGH(3)1' cells transfected with a control plasmid and stimulated with the same GnRH agonist. Transfection of minigenes coding for loop 3i of the M1Ach-muscarinic and the alpha1B-adrenergic (G(q/11)-coupled) receptors resulted in 25-55% inhibition of maximal GnRH-evoked IP turnover. Paradoxically, loop 3i from the M1Ach-muscarinic receptor also maximally inhibited GnRH agonist-stimulated cAMP accumulation and PRL release by 40% (both effects mediated through activation of the Gs protein). Transfection of loop 3i from the D1A -dopamine receptor (coupled to the Gs protein) produced a selective attenuation (40%) in Gs-mediated cellular responses. In contrast, receptor/G protein coupling appeared unaffected by expression of loop 3i domains derived from two receptors coupled to G(i/o) proteins (M2Ach-muscarinic and alpha2A-adrenergic receptors). These data indicate that the third intracellular loop of the rat GnRH-R is involved in receptor G(q/11) protein coupling and/or selectivity, and in the GGH(3)1' cell line, this loop is also involved in signal transduction mediated through the Gs protein pathway.

A rapid, sensitive, and easy-to-perform solid phase insulin radioimmunoassay.

Accurate measurement of basal insulin release in perifusion, perfusion and low-density beta-cell preparations has been difficult with present assays. A simple, competitive, equilibrium, 15-hour insulin assay using 125I-insulin with microtiter plate immobilized antibody, has been developed. This method, a Solid-phase-RadioImmunoAssay (SPRIA), is very sensitive and has a broad useful range (1 - 64 microU/ml). For a test series of 4 standard curves, interassay variation between controls of 1, 4, 16, and 64 microU/ml was +/- 5.2% (SEM) and intra-assay variation over the range of standards between 0.5 to 64 microU/ml was 5.1% (SEM). Nonspecific binding was not significantly different from empty borosilicate culture tubes; 4.0 +/- 0.4 and 3.5 +/- 0.5 counts/minute (mean +/- SEM; n = 54), respectively. This SPRIA can be used with existing gamma-counters, while reducing the radioactive and glass waste presently produced by RIA (test-tubes can be reused). The radioactivity of unused test-tubes was compared against test-tubes used for greater than 10 assays, values were 3.5 +/- 0.5 and 4.4 +/- 0.6 counts/minute mean +/- SEM; n = 54), respectively. Results of an oral glucose tolerance test (oGTT) performed on four male Wistar Furth rats showed a close correlation between SPRIA and RIA insulin values (linear regression, r2 = 0.990). This SPRIA measured plasma insulin levels from a human oGTT with a variation of less than or equal to 3.7% (SEM) between sample triplicates. Standard curves from three commonly measured insulin isoforms (human, rat and porcine) showed a high correlation (multiple linear regression, r2 = 0.998, n = 5 standard curves). In order to determine SPRIA's ability to measure acid extracts, insulin recovery from 2N acetic acid was compared against insulin recovery from Dulbecco's Modified Eagles medium (DME). The insulin recovery from 2N acetic acid was greater than 90% of that achieved with DME. In conclusion, an easy-to-perform assay which is ideal for the rapid quantification of insulin from isolated islets of Langerhans, isolated beta-cells, acetic acid extracts or plasma with greater sensitivity, and less waste than the conventional RIA has been developed.

Regulation of prostaglandin F2alpha-receptor mRNA in human granulosa-luteal cells by human chorionic gonadotrophin and prostaglandin F2alpha.

This study examined the effects of prostaglandin F2alpha (PGF2alpha) and human chorionic gonadotropin (hCG) on the levels of PGF2alpha-receptor (PGF2alpha-R) mRNA and steroidogenesis, in the human granulosa luteal cell (hGLC). Human GLCs collected from patients undergoing in vitro fertilization, were cultured for 24 h, after which cells were exposed to culture media containing either vehicle, hCG (1IU/mL), or hCG plus PGF2alpha (10(-11)-10(-6) M), for a further 24 h. Following the treatment period, media were collected and stored (-20 degrees C) until assayed for progesterone and 17beta-estradiol (estradiol). Immediately following the treatment period, cells were extracted for total RNA. Transcripts for PGF2alpha-R were detected by PCR with two different sets of oligonucleotide primers based on the published human and rat PGF2alpha-R sequences. PCR products were confirmed to be those of PGF2alpha-R by size and by Southern blot hybridization with an internal oligo nucleotide probe. All experiments were performed a minimum of three times, on cells from a minimum of three separate patients. Prostaglandin F2alpha-R mRNA was significantly downregulated, whereas progesterone and estradiol production were significantly stimulated by hCG. Conversely, both low (10(-11)M) and high concentrations (10(-6) M) of PGF2alpha restored PGF2alpha-R mRNA levels to those of the controls, whereas steroidogenesis was significantly inhibited by these conditions. At a concentration of 10(-9)M PGF2alpha-R mRNA was barely detectable. Progesterone and estradiol production were inversely related to PGF2alpha-R levels, since hCG-stimulated progesterone and estradiol production were completely restored in the presence of 10(-9) M PGF2alpha. Messenger RNA levels for the housekeeping gene beta-actin were unaltered by the above treatments. In conclusion, in the human granulosa luteal cell, PGF2alpha-R mRNA levels are inversely related to hCG-stimulated steroidogenesis (which was biphasic in nature). Moreover, in the presence of hCG, PGF2alpha downregulates its receptor mRNA, thus providing a potential form of negative feedback on its own actions, which may be important in rescuing the corpus luteum from PGF2alpha-mediated luteolysis should pregnancy occur.

Stepwise activation of the gonadotropic signal transduction pathway, and the ability of prostaglandin F2alpha to inhibit this activated pathway.

Through selective activation of the gonadotropic signal transduction pathway, we have determined the probable site of the antigonadotropic effects of prostaglandin F2alpha (PGF2alpha) in the human granulosa-luteal cell (hGLC). The gonadotropic signal transduction pathway was activated at the level of the receptor (luteinizing hormone and beta-adrenergic), stimulatory G protein (Gs), adenylate cyclase (AC), and protein kinase A (PKA) by human chorionic gonadotropin (hCG) and isoproterenol (Iso), cholera toxin (CTX), forskolin, and dibutryl cAMP (Db cAMP), respectively. Concomitantly, the ability of PGF2alpha to inhibit progesterone production in response to the activation of this cascade at these different levels was examined. hGLCs were obtained from in vitro fertilization patients and were precultured for 8 d in Medium 199 supplemented with fetal bovine serum (M199; 10% FBS). Following the preculture period, cells were treated with either vehicle or one of the above activators of the gonadotropic pathway, either in the absence or presence of PGF2alpha (in M199; No FBS). Following the treatment period, media were collected and assayed for progesterone by RIA. Prostaglandin F2alpha (10(-6) M) significantly inhibited hCG (1 IU/mL), Iso (10(-5) M), CTX (1 microg/mL), and forskolin- (10(-5) M) stimulated progesterone production. Conversely, PGF2alpha did not inhibit progesterone production stimulated by a saturating concentration of Db cAMP (10(-6) M). The ability of PGF2alpha to inhibit hCG- or CTX-stimulated progesterone production was attenuated by pertussis toxin (PTX; 50 ng/mL). In conclusion, through a pertussis toxin-sensitive G protein, PGF2alpha inhibits progesterone production at a level below AC, and above the activation of PKA by cAMP.

Interaction of prostaglandin F2alpha and gonadotropin-releasing hormone on progesterone and estradiol production in human granulosa-luteal cells.

This study examined the effects of prostaglandin-F2alpha (PGF2alpha), GnRH, and their interactions on steroidogenesis in human granulosa-luteal cells (GLCs). Human GLCs collected from in vitro fertilization patients were cultured for one or eight days, followed by a 24-h treatment period, after which media were collected and radioimmunoassayed for progesterone and estradiol. In the first experiment, GLCs were treated with vehicle, PGF2alpha (10(-9) M), GnRH (10(-6) M), or PGF2alpha plus GnRH, with or without hCG (1 IU/ml). Neither PGF2alpha nor GnRH alone had a significant effect; however, the combination of PGF2alpha plus GnRH significantly stimulated steroidogenesis. Similarly, co-application enhanced the luteolytic effects of PGF2alpha. In a second experiment, PGF2alpha and GnRH concentration-response curves were crossed into a matrix of 49 separate treatments. Responses were plotted in three-dimensions and as two-dimensional "slices" that were analyzed statistically. In the presence of high concentrations of GnRH (10(-6) M), PGF2alpha stimulated progesterone production in a biphasic manner, as middle concentrations significantly stimulated (10(-9) M) whereas low and high concentrations did not. In the presence of middle concentrations of PGF2alpha (10(-9) M), GnRH significantly stimulated progesterone production in a linear concentration-dependent manner. Similar complexities were seen with respect to estradiol response. Thus, in the human GLC, GnRH potentiates the luteolytic effects of PGF2alpha, while it acts as a permissive factor for the luteotropic effects. Furthermore, we have revealed the complex interaction of these hormones using a three-dimensional experimental design.

Interaction of prostaglandin F(2alpha) and prostaglandin E(2) on progesterone production in human granulosa-luteal cells.

This study examined the effects of prostaglandin-F(2alpha) (PGF(2alpha)), prostaglandin-E(2) (PGE(2)) and their interactions on progesterone production in human granulosa-luteal cells (GLCs). Human GLCs collected from in vitro fertilization patients were cultured for 1 (D(1)) or 8 days (D(8)), followed by a 24-hour treatment period, after which media were collected and radioimmunoassayed for progesterone. Seven-point PGF(2alpha) and PGE(2) concentration-response curves were crossed into a matrix of 49 separate treatments. Responses were plotted in three dimensions and as two-dimensional "slices". In D(1) cultured human GLCs neither PGF(2alpha) nor PGE(2) alone had any effect on progesterone production, however two different combinations of these hormones led to at least a 3-fold increase in progesterone production. This stimulation was seen when cells were treated with 10(-6) M PGF(2alpha) plus 10(-9) M PGE(2), and when they were treated with 10(-10) M PGF(2alpha) plus 10(-9) M PGE(2). In D(8) GLCs, PGF(2alpha) stimulated progesterone production maximally at 10(-9) M, while the lowest (10(-11) M) and highest concentrations (10(-6) M) tested were ineffectual. On the contrary, in the presence of high concentrations of PGE(2) (10(-6) to 10(-7) M), PGF(2alpha)-mediated stimulation of progesterone production was attenuated. In a similar fashion to PGF(2alpha), PGE(2) also acted in a luteotrophic manner, although the maximal stimulation of progesterone production was seen at a higher concentration (10(-8) to 10(-7) M). Likewise, PGE(2)-mediated progesterone production was attenuated by the presence of high concentrations of PGF(2alpha) (10(-6) to 10(-7) M). In conclusion, in D(1) human GLCs neither PGF(2alpha) nor PGE(2) alone were luteotrophic, although specific combinations of these hormones were. Conversely, in D(8) GLCs both PGF(2alpha) and PGE(2) stimulated progesterone production in a biphasic manner, while the presence of a high concentration of either of these prostaglandins attenuated the luteotrophic effects of the other. Therefore, PGF(2alpha) and PGE(2) interacted in a concentration-dependent manner, resulting in a multimodal progesterone response, which was easily visualized using three-dimensional plots.