Aspartyl beta -hydroxylase (Asph) and an evolutionarily conserved isoform of Asph missing the catalytic domain share exons with junctin.

The mouse aspartyl beta-hydroxylase gene (Asph, BAH) has been cloned and characterized. The mouse BAH gene spans 200 kilobase pairs of genomic DNA and contains 24 exons. Of three major BAH-related transcripts, the two largest (6,629 and 4,419 base pairs) encode full-length protein and differ only in the use of alternative polyadenylation signals. The smallest BAH-related transcript (2,789 base pairs) uses an alternative 3' terminal exon, resulting in a protein lacking a catalytic domain. Evolutionary conservation of this noncatalytic isoform of BAH (humbug) is demonstrated in mouse, man, and Drosophila. Monoclonal antibody reagents were generated, epitope-mapped, and used to definitively correlate RNA bands on Northern blots with protein species on Western blots. The gene for mouse junctin, a calsequestrin-binding protein, was cloned and characterized and shown to be encoded from the same locus. When expressed in heart tissue, BAH/humbug preferably use the first exon and often the fourth exon of junctin while preserving the reading frame. Thus, three individual genes share common exons and open reading frames and use separate promoters to achieve differential expression, splicing, and function in a variety of tissues. This unusual form of exon sharing suggests that the functions of junctin, BAH, and humbug may be linked.

Cyclooxygenase-2 is essential for normal recovery from 5-fluorouracil-induced myelotoxicity in mice.

Cyclooxygenase (COX) plays a key regulatory role in prostaglandin synthesis. COX-2 is inducible and is the major isoform of inflammatory cells. COX-2-deficient mice were shown to have normal basal hematopoiesis and hematology. We hypothesized that COX-2 induction plays a role in the recovery phase of 5-fluorouracil (5-FU) induced bone marrow injury, because significant macrophage-driven phagocytic removal of necrotic debris and stromal cell reorganization of repopulating marrow occur after 5-FU induction of bone marrow necrosis. Hematologic recovery was markedly delayed with moderately severe leukopenia, thrombocytopenia and reticulocytopenia compared to heterozygotes on day 8 or 12 in Cox-2-/- mice. Mild anemia was present in 5-FU-treated Cox-2-/- and Cox-2+/- mice on days 8 and 12, which was more severe in Cox-2-/- mice. Cox-2-/- mice had markedly decreased bone marrow cell counts per femur and reduced numbers of erythroid and myeloid colony-forming cells compared to heterozygote mice on days 8 and 12 post 5-FU. Histologic examination of 5-FU-treated Cox-2-/- mice revealed a failure to repopulate the intact marrow stroma with hematopoietic cells. Accelerated erythropoiesis following phenylhydrazine-induced hemolytic anemia, however, was comparable between Cox-2-/- and Cox+/- mice, as were induced levels of renal erythropoietin mRNA. COX-2 induction is likely a central event in the accelerated hematopoiesis following myelotoxic injury, because recovery from 5-FU-induced myeloablation is markedly impaired in Cox-2-/- mice but is normal after phenylhydrazine induction of anemia.

Multiple female reproductive failures in cyclooxygenase 2-deficient mice.

Cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of prostaglandins (PGs) and exists in two isoforms, COX-1 and COX-2. In spite of long-standing speculation, definitive roles of PGs in various events of early pregnancy remain elusive. We demonstrate herein that the targeted disruption of COX-2, but not COX-1, in mice produces multiple failures in female reproductive processes that include ovulation, fertilization, implantation, and decidualization. Using multiple approaches, we conclude that these defects are the direct result of target organ-specific COX-2 deficiency but are not the result of deficiency of pituitary gonadotropins or ovarian steroid hormones, or reduced responsiveness of the target organs to their respective hormones.

Suppression of intestinal polyposis in Apc delta716 knockout mice by inhibition of cyclooxygenase 2 (COX-2).

Two cyclooxygenase isozymes catalyze conversion of arachidonic acid to prostaglandin H2: constitutive COX-1 and inducible COX-2. To assess the role of COX-2 in colorectal tumorigenisis, we determined the effects of COX-2 gene (Ptgs2) knockouts and a novel COX-2 inhibitor on Apc delta716 knockout mice, a model of human familial adenomatous polyposis. A Ptgs2 null mutation reduced the number and size of the intestinal polyps dramatically. Furthermore, treating Apc delta716 mice with a novel COX-2 inhibitor reduced the polyp number more significantly than with sulindac, which inhibits both isoenzymes. These results provide direct genetic evidence that COX-2 plays a key role in tumorigenesis and indicate that COX-2-selective inhibitors can be a novel class of therapeutic agents for colorectal polyposis and cancer.

Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II.

Prostaglandins have wide-ranging effects in the body and are thought to be important mediators of inflammation. Cyclooxygenase (COX) plays a key regulatory role in prostaglandin synthesis, and occurs in both constitutive (COX-1) and inducible (COX-2) isoforms. COX-1 is thought to provide cytoprotective effects, whereas COX-2 is both inducible and the major isoform of inflammatory cells. Reduction of prostaglandin production by inhibition of cyclooxygenases appears to be the main mechanism of action of most non-steroidal anti-inflammatory drugs (NSAIDS). Here we present an animal model of COX-2 deficiency that was generated by gene targeting. Defects in null mice correlating with reduced viability included renal alterations, characteristic of renal dysplasia (100% penetrance), and cardiac fibrosis (50% penetrance). Female Cox-2-/- mice were infertile. COX-2 deficiency failed to alter inflammatory responses in several standard models, but striking mitigation of endotoxin-induced hepatocellular cytotoxicity was observed.

Fluorescence in situ hybridization of interphase nuclei isolated from whole blood of transgenic mice.

Homozygosity in transgenic mice is typically confirmed by the time-consuming practice of breeding suspected homozygotes to non-transgenics and looking for 100% transmittance of the transgene to offspring. We have devised a simple reproducible method for isolating white blood cell nuclei from small quantities of mouse whole blood and have utilized fluorescence in situ hybridization to confirm the presence of one (hemizygous) or two (homozygous) copies of the transgene locus in interphase nuclei fixed to microscope slides. This method should have other applications where in situ hybridization of interphase nuclei is desirable.

Flow cytometric analysis of transport activity in lymphocytes electroporated with a fluorescent organic anion dye.

Organic anion transport in polarized epithelia and macrophages has previously been studied by monitoring the efflux of fluorescent organic anion dyes from cells. We adapted this strategy to the study organic anion transport in lymphocytes. Cloned lymphoma cells and normal and activated human T cells were loaded with a membrane-impermeant, organic anion dye (Lucifer Yellow) by electroporation. Dye efflux in lymphocytes was rapid, energy-dependent, and inhibitable by organic anion transporter inhibitors. Dye efflux could not be attributed to the effects of electroporation. In addition, electroporated, dye-loaded T helper cells retained the ability to properly respond to specific antigen. Thus, dye loss occurred in viable, functionally competent cells. These experiments demonstrate that electroporation is an effective means of loading cells with Lucifer Yellow, and that lymphocytes possess organic anion transporters that are functionally similar to those previously described for secretory epithelia and macrophages.

Postreplication labeling of E-leaflet molecules: membrane immunoglobulins localized in sectioned, labeled replicas examined by TEM and HVEM.

Conventional freeze-fracture techniques were combined with immunogold labeling and with plastic embedding and sectioning to analyze the distribution of membrane immunoglobulins (mIgs) and their associated intramembrane particles (IMPs) in E-face replicas of murine B-lymphocyte plasma membranes. Immunogold labels were applied to cells after the process of freeze-fracture and replication. Conventional stereoscopic transmission electron microscopic examination of sectioned, labeled replicas (SLRs) revealed that the gold-labeled mIgs were bound to and localized on the outer leaflets of split and replicated membranes. The gold labels were attached to the external determinants of the mIg molecules, which were retained beneath and contiguous with the replicated E-faces. The mIgs were also localized on the external surface of unreplicated microvilli. In addition, thick sections examined by high-voltage transmission electron microscopy (HVEM) revealed large expanses of replica with well-resolved IMPs. mIgs colocalized with small-diameter (less than 60 A) IMPs in E-face replicas of B-lymphocytes whose mIgs were patched by anti-immunoglobulin. Thus, postreplication E-surface labeling of split and replicated membranes is a high-resolution technique that is suitable for the study of membrane protein distribution in E-face replicas and contiguous nonreplicated tissue.

Effects of estramustine and megestrol on ultrastructure of rat ventral prostate.

The effects of estramustine phosphate on the ultrastructure of the rat ventral prostate closely resemble postcastration events, i.e., a decrease in cell size and secretory activities. The fibromuscular stroma, especially collagen fibers, appear to lose their adherence to adjoining muscle cells and fibroblasts. An infiltration of lymphocytes was noted in dilated regions of the fibromuscular stroma. There was an increase in cellular activity in the ventral prostate of rats treated with megestrol acetate with varying degrees of cellular atrophy and cytoplasmic disorganization. Squamous metaplasia was evident in one experimental group of MA-treated animals. Particles resembling mature C-type viruses were observed in several prostates from control and experimental animals.