Expression of cyclooxygenase-2 in embryonic and fetal tissues during organogenesis and late pregnancy.

BACKGROUND: Cyclooxygenase (COX) catalyzes the committed step in prostaglandin biosynthesis and exists as two related but unique isoforms, COX-1 (constitutive) and COX-2 (inducible). Prostaglandins (PGs) are known to have many important functions in reproduction, such as placentation and decidualization. Studies with the COX-1 and COX-2 knockout mice have demonstrated that COX-2, but not COX-1, is crucial for normal ovulation, implantation, and decidualization, suggesting that COX-2-derived PGs are important during the initial stages of pregnancy. Although the COX-2 knockout mice did not exhibit any abnormalities at birth, relatively little information exists with regard to the expression of COX-2 in the fetus during development. METHODS: In order to understand the role of COX-2 throughout pregnancy, we characterized the cell type and the temporal expression of inducible COX-2 throughout embryonic and fetal development in the rat (n = 22) by immunohistochemistry and in situ hybridization. RESULTS: High levels of COX-2 expression were seen in decidualized uterine tissue on gestation days 7-13 and then in the fetal membranes on gestation days 17-20. Cyclooxygenase-2 expression was not detectable in any tissues from developing embryos during gestation days 7-13, but was observed in the fetal growth period (gestation days 15-20) in the skin, heart, cartilage, and the kidney. CONCLUSIONS: No COX-2 expression was seen in fetal tissues at days 7-13 of gestation, but was seen in various tissues at days 15-17 of gestation. These observations suggest that COX-2 may be important in mid to late pregnancy through an effect on fetal organ growth, but not in the organogenetic phase of fetal development.

Effect of the storage period of paraffin sections on the detection of mRNAs by in situ hybridization.

In this study we evaluated whether storing non-deparaffinized sections can affect the detection of specific mRNAs by radioactive in situ hybridization (ISH). Using a standard ISH protocol, we hybridized serial sections of paraffin blocks stored for different periods of time with (33)P-labeled riboprobes specific for rat Type III collagen and matrix metalloproteinase-2 (MMP-2). Signal intensities were evaluated using a phosphorimager and by blinded microscopic examination. For slides hybridized with the Type III collagen riboprobe, signal intensities measured with the phosphorimager or evaluated by microscopic examination were negatively correlated with the storage period of the sections. For slides hybridized with the MMP-2 riboprobe, differences in signal intensity could be detected, albeit inconsistently, with the phosphorimager, although microscopic examination consistently indicated stronger signals in freshly sectioned slides compared to slides stored for 2 weeks or more. We concluded that it was preferable to use recently prepared sections for trying to locate mRNAs in paraffin-embedded tissues by ISH. In addition, our results suggest that quantifying signal intensity using a phosphorimager is feasible for abundant mRNAs or when large differences in expression are anticipated.(J Histochem Cytochem 49:927-928, 2001)

Novel 6-alkoxypurine 2',3'-dideoxynucleosides as inhibitors of the cytopathic effect of the human immunodeficiency virus.

Twenty-one 6-alkoxypurine 2',3'-dideoxynucleosides were enzymatically synthesized with nucleoside phosphorylases purified from E. coli. Eighteen analogs exhibited anti-HIV-1 activity in MT4 cells. Two analogs, 6-(hexyloxy)-(17) and 6-(heptyloxy)-(18) purine 2',3'-dideoxynucleoside, were as potent as 2',3'-dideoxyinosine (ddI, didanosine, Videx). Although the antiviral activities of 17 and 18 were equivalent, 18 was more cytotoxic. Analogs containing less than four carbons in the 6-alkoxypurine substituent exhibited weak anti-HIV-1 activity. Analogs containing more than seven carbons in the 6-alkoxypurine substituent were too cytotoxic to be effectively evaluated for antiviral activity. Several 6-alkoxypurine 2',3'-dideoxynucleosides were evaluated for substrate activity with calf intestinal adenosine deaminase (ADA). Increasing the carbon chain length of the 6-alkoxypurine substituent decreased the rate of dealkoxylation. The best substrate in this series was 6-methoxypurine 2',3'-dideoxynucleaside (1); however, the rate of dealkoxylation of 100 microM 1 was 0.17% of the rate of deamination of 100 microM 2',3'-dideoxyadenosine. Compound 17, the most potent anti-HIV-1 analog, was not a substrate for ADA. EHNA (erthro-9-(2-hydroxy-3-nonyl)adenine), a potent inhibitor of ADA, had little effect on the antiviral activities of 17 and ddI. In contrast, coformycin, a potent inhibitor of both ADA and AMP deaminase, dramatically decreased the antiviral activity of 17, but not the antiviral activity of ddI. Thus, AMP deaminase appeared to be involved in the anabolism of 17. The pharmacokinetic profile of 17, the most promising analog in this series, was determined in the rat. At least seventeen metabolites of 17, including ddI, were detected in plasma samples. This analog also had poor oral bioavailability.