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.     

A mathematical model and computer simulation study of insulin receptor regulation

A homeomorphic mathematical model of cell surface insulin receptor regulation is developed. The overall structure of the model is based on molecular mechanisms suggested by in vivo and in vitro experimental evidence from many different cell types. Model parameters correspond to cellular processes which are constrained by known boundry value conditions. As an example, computer simulation results are compared with published data from BC3H-1 myocytes in culture. With appropriate parameter choice, this model is able to simulate data from other cell types. Cellular processes which are explicitly represented in the model include: bound and unbound receptor endocytosis, receptor recycling, intracellular receptor degradation, and state-dependent receptor synthesis. Most of these processes are represented as first-order events. Using more complex representations of the model structure with higher order rate constants or saturable pathways does not qualitatively improve simulation results. Simulations are able to reproduce ligand-induced down and up regulation of receptors as well as the initial spontaneous display of surface insulin receptors. To demonstrate the behavior of our model and illustrate its utility for explaining insulin receptor regulation for a variety of conditions, simulations for which experimental data is unavailable for direct comparison are also shown. We believe the structure of our model is sufficient to explain insulin receptor regulation in a wide variety of cell types. In addition our model may aid in understanding the receptor component of insulin resistance (decreased sensitivity or responsiveness to insulin) seen in pathological states such as obesity and diabetes mellitus. Finally, this model may be applicable to the study of the regulation of other polypeptide hormone receptors.     

Cloning and regulation of messenger RNA for mouse apolipoprotein E

A cDNA clone for mouse apolipoprotein E has been identified from a mouse liver cDNA library by a combination of differential colony hybridization and hybrid selection-translation. The identity of the clone was unambiguously established by partial sequencing and comparison with human apolipoprotein E nucleotide and amino acid sequences. In conjunction with an in vitro translation assay for apolipoprotein E, the clone has been used to examine the relative levels of apolipoprotein E mRNA in various tissues of the mouse and the regulation of apolipoprotein E synthesis in response to a diet rich in saturated fat and cholesterol. In the tissues examined, the clone was found to hybridize to a polyadenylated RNA species of approximately 1400 nucleotides. Of the tissues involved in lipoprotein synthesis, liver is very rich (about 1% of total) in apolipoprotein E mRNA while intestine contains only trace amounts. Appreciable levels of active apolipoprotein E mRNA (up to 10% of that in liver) are also detected in peripheral tissues not associated with lipoprotein synthesis, including lung, kidney, spleen, and heart. Thus, extrahepatic apolipoprotein E synthesis may contribute significantly to the levels present in plasma, and a possible function in "reverse cholesterol transport" is considered. When mice were placed on a high lipid diet there was no discernible change in the level of apolipoprotein E mRNA in liver or intestine, although the level of the circulating protein increased about 3-fold. We conclude that in mice the effect of diet on apolipoprotein E levels in blood does not result from induction of mRNA in these tissues.     

Fibroblast growth factor induces beta-amyloid precursor mRNA in glial but not neuronal cultured cells.

Treatment of PC12 and C6 cell cultures with recombinant basic fibroblast growth factor results in approximately a five to ten-fold stimulation of beta-amyloid precursor mRNA in the C6 astrocytoma cell line but only a slight induction of precursor mRNA in the PC-12 neuronal cell line. Stimulation of expression occurred at a hormone concentration of approximately 0.5 to 1 nM and was seen after 2 days. These results suggest that basic fibroblast growth factor may contribute to amyloidosis of Alzheimer's disease.     

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.     

A gene-controlling response of bone marrow progenitor cells to granulocyte-macrophage colony stimulating factors

The production of granulocytes and macrophages from progenitor cells in the bone marrow is controlled, in part, by a family of humoral regulators, termed colony stimulating factors (CSF). We have examined genetic factors controlling this process using in vitro cloning techniques. The inbred mouse strain LP/J showed elevated colony formation (CFU-C) in response to one subtype of CSF (G,M-CSF) compared to other strains of mice examined including the strain C57BL/6J. This variation resulted in a shift to the left of the CFU-C dose-response curve for LP/J. No difference between LP/J and C57BL/6J was seen with another subtype of CSF (CSF-1). Maximal CFU-C response was similar in the two mouse strains with both types of CSF, and mixing experiments with both types of CSF gave the same maximal level of colony formation as the individual CSF. (C57BL/6J X LP/J)F1 progeny exhibited a CFU-C dose-response curve to CSF-2 that was intermediate between the parental types, indicating additive inheritance. Genetic analysis of backcross progeny suggested that the variation in CFU-C response is probably determined by a single primary gene, although the variability of the colony formation assay has complicated interpretation of genetic studies. These results suggest that CSF-1 and G,M-CSF act independently on a single bone marrow progenitor cell population. The properties of the genetic variation for G,M-CSF response are consistent with an alteration in cellular receptors for G,M-CSF.     

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.     

Facilitation and herbivory during restoration of California coastal sage scrub

Using nurse plants to facilitate native plant recruitment in degraded habitats is a common restoration practice across various arid and semiarid environments. Living trees or shrubs are typically considered nurse plants, whereas dead shrubs left in the landscape from prolonged drought are understudied prospective facilitators for native plant recruitment. The interaction between nurse plants and biotic pressures, such as herbivory, on native recruitment is also not well understood in semiarid plant communities. We investigated the effects of facilitation and herbivory on native seedling germination, growth, and survival in the restoration of degraded coastal sage scrub (CSS) habitat. Native shrub seedlings (Artemisia californica and Salvia mellifera) were planted, and native annual species (Amsinckia intermedia, Deinandra fasciculata, Phacelia distans, and Pseudognaphalium californicum) were sown in three Shrub Type treatments (live shrub, dead shrub, and exposed areas), with a nested Cage treatment (no cage and cage) in each Shrub Type treatment. Annual species grew equally well in all Shrub Type treatments; shrub seedlings grew largest in exposed areas. While there was little evidence of facilitation for all species tested, there were strong positive effects of caging on growth and establishment of all species. Caging palatable native species or planting species with anti‐herbivory traits around target plants may be more strategic approaches compared to using nurse plants in restoring degraded CSS after extended drought.     

Phosphorylation of PTP1B at Ser(50) by Akt impairs its ability to dephosphorylate the insulin receptor

PTP1B is a protein tyrosine phosphatase that negatively regulates insulin sensitivity by dephosphorylating the insulin receptor. Akt is a ser/thr kinase effector of insulin signaling that phosphorylates substrates at the consensus motif RXRXXS/T. Interestingly, PTP1B contains this motif (RYRDVS(50)), and wild-type PTP1B (but not mutants with substitutions for Ser(50)) was significantly phosphorylated by Akt in vitro. To determine whether PTP1B is a substrate for Akt in intact cells, NIH-3T3(IR) cells transfected with either wild-type PTP1B or PTP1B-S50A were labeled with [(32)P]-orthophosphate. Insulin stimulation caused a significant increase in phosphorylation of wild-type PTP1B that could be blocked by pretreatment of cells with wortmannin or cotransfection of a dominant inhibitory Akt mutant. Similar results were observed with endogenous PTP1B in untransfected HepG2 cells. Cotransfection of constitutively active Akt caused robust phosphorylation of wild-type PTP1B both in the absence and presence of insulin. By contrast, PTP1B-S50A did not undergo phosphorylation in response to insulin. We tested the functional significance of phosphorylation at Ser(50) by evaluating insulin receptor autophosphorylation in transfected Cos-7 cells. Insulin treatment caused robust receptor autophosphorylation that could be substantially reduced by coexpression of wild-type PTP1B. Similar results were obtained with coexpression of PTP1B-S50A. However, under the same conditions, PTP1B-S50D had an impaired ability to dephosphorylate the insulin receptor. Moreover, cotransfection of constitutively active Akt significantly inhibited the ability of wild-type PTP1B, but not PTP1B-S50A, to dephosphorylate the insulin receptor. We conclude that PTP1B is a novel substrate for Akt and that phosphorylation of PTP1B by Akt at Ser(50) may negatively modulate its phosphatase activity creating a positive feedback mechanism for insulin signaling.