Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.     

Reversal of lovastatin-mediated inhibition of natural killer cell cytotoxicity by interleukin 2

The activation of human natural killer (NK) cell cytotoxicity by interleukin 2 (IL-2) is well established, although the biochemical mechanisms of this stimulation have not yet been fully delineated. Earlier, we reported that treatment of NK cells with an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase such as compactin or lovastatin significantly abrogates the in vitro killing of a susceptible human erythroleukemic cell line and that this inhibition can be completely reversed by 2 hr of exposure to mevalonate (J. Cell. Physiology 139:550-557, 1989). We report here that 24 hr of treatment with IL-2 also reverses lovastatin inhibition of NK cell function. In addition to natural cytotoxicity, IL-2 also restores chemotactic and antibody dependent cellular cytotoxicity functions to lovastatin-treated cells. IL-2 does not stimulate proliferation of these cells during this time period, nor does it affect the phenotypic composition of the NK cell preparations. Although IL-2 was able to reverse the lovastatin-mediated inhibition of every cell function we examined, it had no effect on the inhibition of cholesterol biosynthesis as measured by [3H]acetate incorporation into non-saponifiable lipids, nor did it stimulate HMG CoA reductase activity. These findings support the hypothesis that there is a non-sterol isoprenoid product which is required for NK cell cytotoxicity and chemotaxis. In addition, the data suggest that IL-2 stimulation of NK cells proceeds by an isoprenoid-independent pathway.     

Immunohistochemical study of the developing endocrine pancreas of the opossum (Didelphis virginiana)

Cells immunoreactive for insulin, glucagon, somatostatin, bovine pancreatic polypeptide and 5-hydroxytryptamine are found in the pancreas of the newborn opossum and of all later stages examined. All immunoreactive cell types are present in primary and secondary islets and within elements of the exocrine pancreas. Cells immunoreactive for glucagon, bovine pancreatic polypeptide, somatostatin and 5-hydroxytryptamine generally are confined to the periphery of secondary (intralobular) islets, whereas insulin-immunoreactive cells occupy the central region. Endocrine cells within primary (interlobular) islets are randomly scattered. A small number of pancreatic-polypeptide-immunoreactive cells are reactive for the amine 5-hydroxytryptamine also, but the reverse is not observed. The endocrine pancreas continues to differentiate and develop throughout postnatal life and into adulthood. Little difference was observed between the head and tail regions of the opossum pancreas for the measurements made.     

Use of oligonucleotides to define the site of interstrand cross-links induced by Adriamycin.

It has been known for several years that Adriamycin forms adducts and interstrand cross-links when reacted for long periods of time with bacterial and mammalian DNA in vitro, with the cross-link being restricted to 2 bp elements containing GpC sequences. The self-complementary 20mer deoxyoligonucleotide TA4T4GCA4T4A has been used in this study as a model of the apparent G-G cross-linking site at GpC sequences. The rate of formation of cross-links, as well as the dependence on both Adriamycin and Fe(III) concentration, were similar with this oligonucleotide as compared with calf thymus DNA. The cross-linking was demonstrated on both denaturing and non-denaturing sequencing gels. The half-life of the G-G cross-link was 40 h, consistent with that implied with high molecular weight, heterogeneous sequence DNA. Exonuclease III digests of adducts formed with 20mer deoxyoligonucleotides containing single, central G-G, G-I and I-I potential cross-links revealed that a guanine residue is required at both ends of the cross-link. No cross-linking was observed with a similar oligonucleotide containing only a single central (G.C) bp.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Cholesterol accumulation caused by low density lipoprotein receptor deficiency or a cholesterol-rich diet results in ectopic bone formation during experimental osteoarthritis

Introduction

Osteoarthritis (OA) is associated with the metabolic syndrome, however the underlying mechanisms remain unclear. We investigated whether low density lipoprotein (LDL) accumulation leads to increased LDL uptake by synovial macrophages and affects synovial activation, cartilage destruction and enthesophyte/osteophyte formation during experimental OA in mice.

Methods

LDL receptor deficient (LDLr^−/−) mice and wild type (WT) controls received a cholesterol-rich or control diet for 120 days. Experimental OA was induced by intra-articular injection of collagenase twelve weeks after start of the diet. OA knee joints and synovial wash-outs were analyzed for OA-related changes. Murine bone marrow derived macrophages were stimulated with oxidized LDL (oxLDL), whereupon growth factor presence and gene expression were analyzed.

Results

A cholesterol-rich diet increased apolipoprotein B (ApoB) accumulation in synovial macrophages. Although increased LDL levels did not enhance thickening of the synovial lining, S100A8 expression within macrophages was increased in WT mice after receiving a cholesterol-rich diet, reflecting an elevated activation status. Both a cholesterol-rich diet and LDLr deficiency had no effect on cartilage damage; in contrast, ectopic bone formation was increased within joint ligaments (fold increase 6.7 and 6.1, respectively). Moreover, increased osteophyte size was found at the margins of the tibial plateau (4.4 fold increase after a cholesterol-rich diet and 5.3 fold increase in LDLr^−/− mice). Synovial wash-outs of LDLr^−/− mice and supernatants of macrophages stimulated with oxLDL led to increased transforming growth factor-beta (TGF-β) signaling compared to controls.

Conclusions

LDL accumulation within synovial lining cells leads to increased activation of synovium and osteophyte formation in experimental OA. OxLDL uptake by macrophages activates growth factors of the TGF-superfamily.     

Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.     

The interaction between thymine DNA glycosylase and nuclear receptor coactivator 3 is required for the transcriptional activation of nuclear hormone receptors

The cellular counterpart of the “soluble” guanylyl cyclase found in tissue homogenates over 30 years ago is now recognized as the physiological receptor for nitric oxide (NO). The ligand-binding site is a prosthetic haem group that, when occupied by NO, induces a conformational change in the protein that propagates to the catalytic site, triggering conversion of GTP into cGMP. This review focuses on recent research that takes this basic information forward to the beginnings of a quantitative depiction of NO signal transduction, analogous to that achieved for other major transmitters. At its foundation is an explicit enzyme-linked receptor mechanism for NO-activated guanylyl cyclase that replicates all its main properties. In cells, NO signal transduction is subject to additional, activity-dependent modifications, notably through receptor desensitization and changes in the activity of cGMP-hydrolyzing phosphodiesterases. The measurement of these parameters under varying conditions in rat platelets has made it possible to formulate a cellular model of NO-cGMP signaling. The model helps explain cellular responses to NO and their modification by therapeutic agents acting on the guanylyl cyclase or phosphodiesterase limbs of the pathway.