Comparison of rain gauge and radar data as input to an urban rainfall-runoff model.

This paper presents an application of radar data (DX-product of the German Weather Service) with a high resolution in space (1 degree x 1 km) and time (delta t = 5 minutes) in urban hydrology. The radar data and data of rain gauges with different locations in the test catchment were compared concerning their suitability as input into an urban rainfall-runoff model. In order to evaluate the accuracy of model simulation results, five evaluation criteria have been specified which are relevant for an efficient management of sewer systems and wastewater treatment plants. The results demonstrate that radar data should be used in urban hydrology if distances > 4 km between rain gauge and catchment exist and for catchments with a density of rain gauges smaller than 1 rain gauge per 16 km2.

Tissue inhibitor of matrix metalloproteinase-3 expression in the mouse uterus during implantation and artificially induced decidualization.

During implantation in mice, tissue inhibitor of matrix metalloproteinases-3 is believed to play a key role in inhibiting matrix metalloproteinase activity associated with embryo invasion and tissue remodeling. The first objective of this study was to quantitatively compare the steady-state mRNA levels of tissue inhibitors of matrix metalloproteinases between segments of the mouse uterus undergoing decidualization compared to those that are not during early pregnancy plus oil-induced decidualization. Steady-state tissue inhibitor of metalloproteinase-3 mRNA levels were significantly greater in implantation compared to interimplantation areas on days 6 and 7 of pregnancy and in stimulated compared to nonstimulated uterine horns at 48 and 72 hr after artificial induction of decidualization. Steady-state tissue inhibitor of metalloproteinase-1 mRNA levels were significantly greater in implantation compared to interimplantation areas on days 5-8 of pregnancy and in stimulated compared to nonstimulated uterine horns at 24, 48, and 72 hr after oil stimulation. Therefore, the steady-state mRNA levels of tissue inhibitors of metalloproteinase-1 and -3 increased in the uterus during decidualization. The second objective of this study was to determine if transforming growth factor-beta1 influences tissue inhibitors of metalloproteinase mRNA concentrations in mouse endometrial stromal cells. As determined by Northern blot analyses, transforming growth factor beta1 significantly increased tissue inhibitors of matrix metalloproteinases-1 and -3 mRNA levels in cultured mouse endometrial stromal cells isolated from uteri sensitized for decidualization. On the other hand, interleukin-1, epidermal growth factor, and leukemia inhibitory factor had no effect. The results of this study further characterize the tissue inhibitor of metalloproteinase expression in the uterus during implantation and artificially induced decidualization and the potential control of their expression in the stroma by transforming growth factor.

Electrospray ionization mass spectrometry-based genotyping: an approach for identification of single nucleotide polymorphisms.

The high frequency of single nucleotide polymorphisms (SNPs) in the human genome makes them ideal genetic markers for mapping, diagnosing disease-related alleles, and identifying SNPs that contribute to drug response differences between individuals. Here we report a novel assay utilizing a single nucleotide primer extension (SNuPE) and electrospray ionization mass spectrometry (ESI-MS) detection for the analysis of SNPs. In contrast to most SNuPE genotyping technologies that detect the extended primer product, the novel Survivor assay detects the unreacted dideoxynucleotides (ddNTPs) remaining or surviving in solution following a SNuPE. This assay involves a simple analysis of the same four ddNTP analytes, regardless of the SNP being investigated, and either single or double-stranded DNA can be used to genotype a SNP, without any labeling requirements of the ddNTPs or oligonucleotide primers. We have tested and blindly validated the Survivor assay by genotyping the C/T SNP at -857 of the human TNFalpha promoter gene. The results obtained are in agreement with the control sequencing data. The results demonstrate that the homogeneous Survivor assay with ESI-MS detection offers advantages in simplicity, accuracy, specificity, and sensitivity. Additional advantages of the method include enhanced hybridization efficiencies in this solution-phase assay and the elimination of immobilized primers for the isolation of single-stranded DNA. With a one-well reaction and an automation platform being developed, the Survivor assay provides a powerful new tool for large-scale SNP analysis and screening.

A four-column parallel chromatography system for isocratic or gradient LC/MS analyses.

A novel approach to parallel liquid chromatography/ tandem mass spectrometry (LC/MS/MS) analyses for pharmacokinetic assays and for similar quantitative applications is presented. Modest modifications render a conventional LC/MS system capable of analyzing samples in parallel. These modifications involve the simple incorporation of three valves and four LC columns into a conventional system composed of one binary LC pumping system, one autosampler, and one mass spectrometer. An increase in sample throughput is achieved by staggering injections onto the four columns, allowing the mass spectrometer to continuously analyze the chromatographic window of interest Using this approach, the optimized run time is slightly greater than the sum of the widths of the desired peaks. This parallel chromatography unit can operate under both gradient and isocratic LC conditions. To demonstrate the utility of the system, atorvastatin, five of its metabolites, and their deuterated internal standards (IS) were analyzed using gradient elution chromatography conditions. The results from a prestudy assay evaluation (PSAE) tray of standards and quality control (QC) samples from extracted spiked human plasma are presented. The relative standard deviation and the accuracy of the QC samples did not exceed 8.1% and 9.6%, respectively, which is well within the acceptance criteria of the pharmaceutical industry. For this particular analysis, the parallel chromatography system decreased the overall run time from 4.5 to 1.65 min and, therefore, increased the overall throughput by a factor of 2.7 in comparison to a conventional LC/MS/MS analytical method.

Pleiotrophin messenger ribonucleic acid levels increase in mouse endometrial stromal cells during decidualization.

In mice (and other species) fibroblast-like endometrial stromal cells differentiate into large decidual cells during early pregnancy. These decidual cells, which play an important function during the process of embryo implantation, eventually die or form the maternal component of the placenta. In the current study, with the use of subtractive hybridization methods and Northern blot analysis, we found that steady-state pleiotrophin transcript levels are increased in uterine horns undergoing artificially induced decidualization compared to control horns. Steady-state pleiotrophin transcript levels were significantly (P< 0.01) greater in uterine horns undergoing decidualization compared to the control horn at 48 h and 72 h, but not 24 h, after the application of the deciduogenic stimulus to appropriately sensitized uteri. This increase in pleiotrophin transcript levels was localized to the endometrial stromal cells undergoing decidualization, as determined by in situhybridization. Finally, we also determined if pleiotrophin transcript levels were greater in implantation segments compared to inter-implantation segments of the uterus during early pregnancy. Steady-state pleiotrophin transcript levels were significantly (P< 0.01) greater in implantation compared to inter-implantation segments on day 6 to 8, but not 5 (day 1 = vaginal plug), of pregnancy. In conclusion, pleiotrophin transcript levels increase in the endometrial stromal cells during decidualization suggesting that it might play a role in the process.

Increased expression of a novel heat shock protein transcript in the mouse uterus during decidualization and in response to progesterone.

The objective of the present study was to identify and characterize transcripts whose levels are increased in the mouse uterus during decidualization. Using the method of suppression subtractive hybridization, we identified a novel transcript. This transcript contained a potential open reading frame that coded for a 196-amino-acid protein that shows homologies to the heat shock protein 20 family of genes. This transcript was expressed in several adult tissues and in the embryo. Its steady-state level was significantly greater in implantation segments of the uterus compared to nonimplantation segments. Furthermore, the steady-state levels of this novel transcript were significantly greater in uterine horns undergoing artificially induced decidualization compared to control contralateral horns. Using in situ hybridization methods, signals for the transcript were localized to the endometrial stromal cells that were undergoing decidualization. Finally, we found that progesterone caused a significant increase in the steady-state level of this novel transcript in the uterus when administered to ovariectomized mice. In the presence of estradiol-17 beta, this effect was significantly reduced. In conclusion, we have identified a novel transcript of a potential heat shock protein whose level is significantly increased in the uterus during decidualization and in response to progesterone.

Expression of matrix metalloproteinases 2 and 9 in the mouse uterus during implantation and oil-induced decidualization.

During implantation, matrix metalloproteinases are believed to play roles in the tissue remodelling that accompanies decidualization in the endometrium and in embryo invasion. The objective of this study was to characterize further the expression of matrix metalloproteinases 2 and 9 in the mouse uterus during early pregnancy and oil-induced decidualization. mRNA encoding matrix metalloproteinase 2 was detected in pregnant uteri and uteri undergoing oil-induced decidualization by northern blot analyses. The steady-state concentrations of mRNA encoding matrix metalloproteinase 2 did not change significantly in implantation compared with inter-implantation areas on days 5-8 of pregnancy but were significantly lower in stimulated compared with non-stimulated uterine horns during artificially induced decidualization. mRNA encoding matrix metalloproteinase 9 was also detected in uteri undergoing oil-induced decidualization but not in pregnant uteri. Its concentration was significantly greater in uterine horns undergoing oil-induced decidualization compared with control horns. Immunoreactive matrix metalloproteinases 2 and 9 were detected in the uterus during early pregnancy and oil-induced decidualization by immunohistochemistry, localized to the endometrial stroma, but the staining progressively became weaker and was absent in areas that had undergone decidualization. By day 8 of pregnancy and 72 h after the induction of decidualization, matrix metalloproteinase 2 and 9 proteins remained mainly in the region of non-decidualized stromal cells adjacent to the myometrium. In implantation segments, they were also localized to the region of the trophoblast giant cells. The second objective of the present study was to determine whether endometrial stromal cells isolated from uteri sensitized for decidualization express matrix metalloproteinases 2 and 9. Northern blot analyses and gelatin zymography showed that these cultured cells expressed matrix metalloproteinase 2 and 9, and that transforming growth factor beta1 significantly increased matrix metalloproteinase 9 expression. The results of the present study further characterize matrix metalloproteinases 2 and 9 expression in the uterus during implantation and artificially induced decidualization.

Regulation of cytosolic phospholipase A2 in rat endometrial stromal cells: the role of epidermal growth factor.

The effect of epidermal growth factor on the levels of cytosolic phospholipase A2 mRNA and protein in cultured rat endometrial stromal cells isolated from uteri sensitized for the decidual cell reaction was examined. Treatment with epidermal growth factor increased the steady-state cytosolic phospholipase A2 mRNA and protein levels as demonstrated by Northern and Western blot analyses, respectively. Immunocytochemical analysis demonstrated an increase of cytosolic phospholipase A2 protein in most cells, as opposed to a small subpopulation of cells in culture. These results show that epidermal growth factor causes an increase in steady-state cytosolic phospholipase A2 mRNA and protein levels in rat endometrial stromal cells from uteri sensitized for the decidual cell reaction. Epidermal growth factor receptor ligands may regulate cytosolic phospholipase A2 and thus prostaglandin production in the endometrial stromal cells during implantation.

Changes in the relative abundance of various housekeeping gene transcripts in in vitro-produced early bovine embryos.

The relative abundances of transcripts of different origins and housekeeping functions were measured by Northern blot analysis of RNA samples derived from in vitro-matured oocytes and in vitro-produced bovine embryos at selected stages of early development. The gene products studied included: two mitochondrial transcripts, 12S rRNA and cytochrome b mRNA; two RNAs involved in the processing of other RNAs, U2 and U3 snRNA; and two nuclear-derived transcripts, beta-actin mRNA and histone H3 mRNA. Overall, the RNA levels for the various genes studied remained constant or decreased slightly from the mature oocyte to the 6- to 8-cell or morula stage and were greatly increased in blastocysts. Differences were observed in the degree to which the RNA levels increased and in the timing of the increase. For 12S rRNA, a major increase was not observed until the blastocyst stage where levels increased 7.1 times the amount detected in morulae. Cytochrome b mRNA levels started to increase at the 6- to 8-cell stage and reached levels in blastocysts that were 20 times more than the cytochrome b mRNA level in 2- to 4-cell embryos. U2 snRNA levels did not increase until the blastocyst stage where levels were 6.4 times the amount found in morulae. U3 snRNA and beta-actin mRNA levels started to increase at the morula stage and blastocysts contained 118 and 110 times more U3 snRNA and beta-actin mRNA, respectively, than 6- to 8-cell embryos. However, blastocysts contained only two times the amount of histone H3 mRNA present in 6- to 8-cell embryos.

Changes in ribosomal ribonucleic acid content within in vitro-produced bovine embryos.

The abundance of 28S, 18S, and 5S rRNA was measured by Northern blot techniques applied to RNA samples extracted from bovine oocytes and preattachment embryos produced by in vitro procedures. Total RNA content was estimated by comparing the intensity of hybridization signals of 28S and 18S rRNA probes to embryo RNA samples and to standard curves generated from bovine ovary or bovine oviduct cell RNA. RNA content declined from the oocyte to the morula stage (2.4 +/- 0.3 ng/oocyte, 1.7 +/- 0.5 ng/1-cell embryo, 2.2 +/- 0.9 ng/2- to 4-cell embryo, 0.8 +/- 0.2 ng/6- to 8-cell embryo, and 0.7 +/- 0.2 ng/morula). A marked increase in RNA content, based on levels of hybridization to 28S and 18S rRNA, was observed in blastocysts, in which values averaged 5.3 +/- 0.6 ng/embryo. On a relative basis, 5S rRNA abundance followed a pattern similar to that of 28S and 18S rRNA across the early development period to the blastocyst stage.

Murine tissue inhibitor of metalloproteinases-4 (Timp-4): cDNA isolation and expression in adult mouse tissues.

We have isolated cDNA clones corresponding to a new member of the murine tissue inhibitor of metalloproteinase (TIMP) family, designated Timp-4. The nucleotide sequence predicts a protein of 22,609 Da that contains the characteristic 12 cysteine TIMP signature. TIMP-4 is more closely related to TIMP-2 and TIMP-3 than to TIMP-1 (48%, 45% and 38% identity, respectively). Analysis of Timp-4 mRNA expression in adult mouse tissues indicated a 1.2 kb transcript in brain, heart, ovary and skeletal muscle. This pattern of expression distinguishes Timp-4 from other Timps, suggesting that the TIMP-4 protein may be an important tissue-specific regulator of extracellular matrix remodelling.

Matrix metalloproteinase-9 maps to the distal end of chromosome 2 in the mouse.

The activity and expression of matrix metalloproteinase-9/gelatinase B (MMP-9), an enzyme implicated in the implantation process in mice, was investigated in normal and parthenogenetic blastocyst outgrowths. Conditioned media from parthenogenetic blastocysts after 4 days of culture had reduced levels of MMP-9 activity compared to conditioned medium from normal outgrowths. Levels of MMP-9 mRNA assayed by reverse transcription-polymerase chain reaction methods were also reduced in parthenogenetic blastocysts compared to normal outgrowths. Genetic mapping studies showed that Mmp9 maps to the distal end of chromosome 2 near the proximal boundary of a region affected by genomic imprinting. Both parental alleles of Mmp9, however, are expressed in 11.5-day embryos derived from interspecific crosses of Mus musculus and Mus spretus. Thus, loss of MMP-9 activity in parthenogenetic blastocysts does not appear to be due to imprinting but, rather, due to a defect of trophoblast giant cell proliferation and differentiation.

Expression of proteinases and proteinase inhibitors during embryo-uterine contact in the pig.

Implantation in pigs is noninvasive and characterized by interdigitation of embryonic and endometrial epithelial cell processes. However, when pig embryos are transferred to ectopic sites, trophoblast becomes invasive. The objective of this study was to evaluate expression of proteinases and proteinase inhibitors in pig embryos and uteri at the time of endometrial attachment. RNA was extracted from Day 15.75 pig embryos and uteri and reverse transcribed, and cDNA was amplified by polymerase chain reactions using primers specific for urokinase-type plasminogen activator (uPA), matrix metalloproteinases-2 and -9 (MMP-2 and -9), and tissue inhibitors of MMP-1, -2, and -3 (TIMP-1, -2, and -3). Localization of transcripts for the genes of interest in embryos and uteri was performed using in situ hybridization with antisense riboprobes. Day 15.75 pig embryos and uteri expressed transcripts for uPA, MMP-2 and -9, and TIMP-1, -2, and -3. In situ hybridization revealed weak expression of uPA in the trophectoderm and moderate expression in the adjacent extraembryonic endoderm. TIMP-1 transcripts were abundant in extraembryonic endoderm and scattered throughout the trophectoderm. TIMP-2 appeared to be expressed in all cells of the embryo. TIMP-3 expression was observed in the trophectoderm and, to a lesser extent, in the extraembryonic endoderm. Specific localization of MMP-2 and -9 transcripts above background was not observed by in situ hybridization in either embryos or uterus. Uterine expression of uPA and TIMP-1, -2 and -3 was localized to the endometrial stroma. Transcripts of these genes were not observed in either the luminal or glandular endometrial epithelium. These results suggest that pig embryos and uteri express a wide array of proteinases and proteinase inhibitors during the period of uterine association. The abundant expression of proteinases and proteinase inhibitors during the period of uterine association. The abundant expression of TIMP in pig embryos may partially explain the absence of invasive implantation in this species in contrast to implantation typified by rodents and primates.

Tissue inhibitor of metalloproteinases-3 is the major metalloproteinase inhibitor in the decidualizing murine uterus.

Embryo implantation in the mouse is a highly orchestrated process, a key aspect of which is the invasion of trophoblast cells of the blastocyst into the maternal uterine endometrium. Invasion is facilitated via proteinases expressed by trophoblast cells and balanced by expression of inhibitors of proteinases in the maternal decidua. The predominant proteinase expressed by trophectodermal derivatives of the implanting mouse embryo is matrix metalloproteinase-9 (MMP-9; gelatinase B). Using in situ hybridization, transcripts for MMP-9 were detected in trophoblast cells of the embryo from the earliest stage of decidual formation (day 6.0) examined. MMP-9 transcripts were localized to trophoblast giant cells at the periphery of the embryo at the egg cylinder stage (day 7.0). By the neural-fold stage (day 8.5), expression was restricted to giant cells adjacent to the maternal side of the developing placenta, and by day 9.5 few MMP-9-positive cells remained. The major tissue inhibitor of metalloproteinases (TIMP) produced during this period was TIMP-3. Transcripts encoding TIMP-3 were detected from day 6.0-7.0 in the maternal decidua immediately adjacent to embryonic cells expressing MMP-9. The intensity of TIMP-3 expression in later-stage embryos declined in parallel with MMP-9 expression. Maternal TIMP-3 expression also occurred in the absence of embryonic MMP-9 expression in decidual reactions induced by parthenogenetic embryos (where MMP-9 positive cells were not detected) or in oil-induced deciduomas. These results support the hypothesis that MMP-9 is an important mediator of cellular invasiveness during embryo implantation, and that TIMP-3 serves as a regulator within the uterus to restrict invasion to the site of implantation.

Regulation of urokinase plasminogen activator production in implanting mouse embryo: effect of embryo interaction with extracellular matrix.

Embryo implantation in the mouse is an invasive process and requires the action of proteinases, including plasminogen activator (PA) and metalloproteinases. After the implanting embryo establishes close contact with the endometrium, the invasion process begins, at least in part, through interactions of the embryo with the extracellular matrix in the endometrium. This study determined whether embryo interaction with extracellular matrix components would affect the secretion of PA in vitro. PA in vitro. Mouse embryos were collected from the uterus on Day 3.5 of development, just before implantation, and were cultured dishes precoated with bovine serum, plasma fibronectin, or BSA (control). Embryos cultured on serum- or fibronectin-coated dishes secretes significantly more PA than those cultured on BSA. The effect of fibronectin was inhibited by hexapeptides that contained the integrin-recognizing Arg-Gly-Asp sequence. This indicates that the action of fibronectin in enhancing PA secretion is mediated through its receptor (integrins) in the embryo. Fibronectin fragments reproduced the effect of the whole fibronectin molecule, suggesting that the clustering of integrins by specific ligands is responsible, at least in part, for the increase PA secretion. The increase in PA secretion was a specific response to fibronectin rather than a reflection of increased total protein secretion, and was at least partially a result of the increased steady-state level of PA mRNA in the cultured embryos. Laminin was as effective as fibronectin in promoting PA secretion. Epidermal growth factor increased PA secretion, probably by promoting the interaction of the embryos with the extracellular matrix. In summary, our findings indicate that the interactions of the implanting embryos with their extracellular matrix may regulate trophoblast invasion by controlling PA secretion.

Expression of Oct-4 during differentiation of murine F9 cells.

Oct-4 is a transcription factor that shares a common structural motif with members of the POU family. The mRNA for Oct-4 is found in growing oocytes and in totipotent or pluripotent cells of the early mouse embryo. Oct-4 is down-regulated in embryos during differentiation events associated with blastocyst implantation and gastrulation. Oct-4 gene expression is also down-regulated when murine embryonic stem cells or embryonal carcinoma cells are induced to differentiate in the presence of retinoic acid. A polyclonal antibody that can recognize a unique peptide sequence in the C-terminus of mouse Oct-4 has been prepared. It specifically recognizes Oct-4 protein as tested by Western blots and gel mobility shift assays. This antibody has been used to measure Oct-4 protein levels during retinoic acid induced differentiation of F9 embryonal carcinoma cells, It was observed that Oct-4 protein was abundant in undifferentiated F9 cells but decreased to levels below detection as the cells differentiated, consistent with changes in levels of expression in early embryos.

Insulin-like growth factor-I (IGF-I) colocalizes with IGF-binding proteins (IGFBPs) in mouse and rat ovary.

The cellular localization of insulin-like growth factor (IGF)-I mRNA, IGF-I peptide, and IGF-binding proteins (IGFBPs) was examined in mouse and rat ovaries through use ofin situ hybridization and immunohistochemical methods. IGF-I mRNA was found to be most abundant in granulosa cells, although lower levels were also detected in cells of the theca interna, stroma, and corpus luteum. In contrast, IGF-I immunoreactivity was undetectable or low in granulosa cells, weak and variable in oocytes, high in theca interna and the corpus luteum, and highest in the stroma. Antibodies directed against IGFBP-2, 3, and 5 yielded similar patterns of immunoreactivity to that observed for IGF-I peptide. The results indicate that IGF-I is synthesized in ovarian follicles, and that IGF-I of ovarian or systemic origin becomes localized to sites containing IGFBPs in the ovary.