His-311 and Arg-559 are key residues involved in fatty acid oxygenation in pathogen-inducible oxygenase

Pathogen-inducible oxygenase (PIOX) oxygenates fatty acids into 2R-hydroperoxides. PIOX belongs to the fatty acid alpha-dioxygenase family, which exhibits homology to cyclooxygenase enzymes (COX-1 and COX-2). Although these enzymes share common catalytic features, including the use of a tyrosine radical during catalysis, little is known about other residues involved in the dioxygenase reaction of PIOX. We generated a model of linoleic acid (LA) bound to PIOX based on computational sequence alignment and secondary structure predictions with COX-1 and experimental observations that governed the placement of carbon-2 of LA below the catalytic Tyr-379. Examination of the model identified His-311, Arg-558, and Arg-559 as potential molecular determinants of the dioxygenase reaction. Substitutions at His-311 and Arg-559 resulted in mutant constructs that retained virtually no oxygenase activity, whereas substitutions of Arg-558 caused only moderate decreases in activity. Arg-559 mutant constructs exhibited increases of greater than 140-fold in K(m), whereas no substantial change in K(m) was observed for His-311 or Arg-558 mutant constructs. Thermal shift assays used to measure ligand binding affinity show that the binding of LA is significantly reduced in a Y379F/R559A mutant construct compared with that observed for Y379F/R558A construct. Although Oryza sativa PIOX exhibited oxygenase activity against a variety of 14-20-carbon fatty acids, the enzyme did not oxygenate substrates containing modifications at the carboxylate, carbon-1, or carbon-2. Taken together, these data suggest that Arg-559 is required for high affinity binding of substrates to PIOX, whereas His-311 is involved in optimally aligning carbon-2 below Tyr-379 for catalysis.     

In vitro isolation and establishment of new embryonal cell lines from rat nephroblastoma

Summary Two mixed cell lines designated REN-1 and REN-2 were established successfully in continuous in vitro culture from subcutaneously propagated transplant tissue derived from a nephroblastoma which had occurred spontaneously in an Nb hooded rat. In monolayer culture the lines consisted of clumps, islands, and cords of densely crowded, small basophilic cells of epithelioid character, together with mesenchymelike cells occupying the intervening spaces. The proportion of epithelioid cells to mesenchyme could be enhanced by high seeding densities and the intermittent application ofcishydroxyproline to the medium. A third cell form with the appearance of more mature epithelium was observed as a later development in one of the monolayer cultures (REN-1). This larger epithelial cell and a homogeneous mesenchymal population were isolated as cloned cell lines from REN-1 (REN-1-C/2 and REN-1-C/1, respectively), but attempts to clone the dominant basophilic epithelioid cell were not successful. Light and electron microscopy indicated the small, basophilic epithelioid cells to be morphologically consistent with the undifferentiated embryonal blast cells of the parent tumor. They were a distinctive population unlike known malignant cell lines representative of chemically transformed rat kidney mesenchyme and epithelium. The mesenchymelike cells present in the uncloned cell lines, and cloned in REN-1-C/1, were fibroblasts by ultrastructural criteria and therefore distinct from the epithelioid moiety. Ultrastructurally the larger epithelium cloned in REN-1-C/2 displayed the features of differentiated renal epithelium. Subcutaneous transplantation in syngeneic and allogeneic recipients showed the uncloned parent cell lines containing the basophilic epithelioid population to be highly tumorigenic, producing rapidly growing tumors that were unequivocal nephoblastomas. The mesenchymal clone, REN-1-C/1, was also tumorigenic but, consistent with its fibroblastic nature, produced only fibrosarcomas on transplantation. The mature epithelial clone, REN-1-C/2, transplanted as an anaplastic carcinoma with limited tubule formation. Because of their distinctive morphology and growth behavior, and their ability to proliferate into nephroblastomas on transplantation, the dominant population of basophilic epithelioid cells in the parent uncloned cell lines is considered to represent neoplastic kidney cells of undifferentiated embryonal type. The possible host origin of the mesenchymal population from the supporting stroma of the original transplantation tumor is suggested in discussion. This investigation was supported by research grants CA-24216 and CA-12227 awarded by the National Cancer Institute, Department of Health and Human Services, and in part by the National Cancer Institute of Canada.     

Glutamate Receptor Agonists Stimulate Nitric Oxide Synthase in Primary Cultures of Cerebellar Granule Cells

The glutamate receptor agonist N-methyl-D-aspartate (NMDA) stimulated a rapid, extracellular Ca(2+)-dependent conversion of [3H]arginine to [3H]citrulline in primary cultures of cerebellar granule cells, indicating receptor-mediated activation of nitric oxide (NO) synthase. The NMDA-induced formation of [3H]citrulline reached a plateau within 10 min. Subsequent addition of unlabeled L-arginine resulted in the disappearance of 3H from the citrulline pool, indicating a persistent activation of NO synthase after NMDA receptor stimulation. Glutamate, NMDA, and kainate, but not quisqualate, stimulated both the conversion of [3H]arginine to [3H]citrulline and cyclic GMP accumulation in a dose-dependent manner. Glutamate and NMDA showed similar potencies for the stimulation of [3H]citrulline formation and cyclic GMP synthesis, respectively, whereas kainate was more potent at inducing cyclic GMP accumulation than at stimulating [3H]citrulline formation. Both the [3H]arginine to [3H]citrulline conversion and cyclic GMP synthesis stimulated by NMDA were inhibited by the NMDA receptor antagonist MK-801 and by the inhibitors of NO synthase, NG-monomethyl-L-arginine (MeArg) and NG-nitro-L-arginine (NOArg). However, MeArg, in contrast to NOArg, also potently inhibited [3H]arginine uptake. Kainate (300 microM) stimulated 45Ca2+ influx to the same extent as 100 microM NMDA, but stimulated [3H]citrulline formation to a much lesser extent, which suggests that NO synthase is localized in subcellular compartments where the Ca2+ concentration is regulated mainly by the NMDA receptor.     

N-Acetylaspartylglutamate Selectively Activates mGluR3 Receptors in Transfected Cells

Abstract: In previous studies, we demonstrated that the neuropeptide, N -acetylaspartylglutamate (NAAG), meets the traditional criteria for a neurotransmitter and selectively activates metabotropic glutamate receptor mGluR2 or mGluR3 in cultured cerebellar granule cells and glia. Sequence homology and pharmacological data suggest that these two receptors are highly related structurally and functionally. To define more rigorously the receptor specificity of NAAG, cloned rat cDNAs for mGluR1–6 were transiently or stably transfected into Chinese hamster ovary cells and human embryonic kidney cells and assayed for their second messenger responses to the two endogenous neurotransmitters, glutamate and NAAG, as well as to metabotropic receptor agonists, trans -1-aminocyclopentane-1,3-dicarboxylate ( trans -ACPD) and l -2-amino-4-phosphonobutyrate ( l -AP4). Despite the high degree of relatedness of mGluR2 and mGluR3, NAAG selectively activated the mGluR3 receptor. NAAG activated neither mGluR2 nor mGluR1, mGluR4, mGluR5, or mGluR6. The mGluR agonist, trans -ACPD, activated each of the transfected receptors, whereas l -AP4 activated mGluR4 and mGluR6, consistent with the published selectivity of these agonists. Hybrid cDNA constructs of the extracellular domains of mGluR2 and mGluR3 were independently fused with the transmembrane and cytoplasmic domain of mGluR1a. This latter receptor domain is coupled to phosphoinositol turnover, and its activation increases intracellular calcium. The cells transfected with these chimeric receptors responded to activation by glutamate and trans -ACPD with increases in intracellular calcium. NAAG activated the chimeric receptor that contained the extracellular domain of mGluR3 and did not activate the mGluR2 chimera.     

Characteristics of glottis-induced turbulence in oscillatory flow: an empirical investigation.

Turbulence inducement from the glottis was scrutinized by employing an idealized model of the larynx and trachea for oscillatory flow conditions. The characterization of turbulence was achieved with the two-component velocity measurements of split-film probe anemometry and with the flow visualization of a smoke-wire technique. The apertures of two different (triangular and circular) shapes were utilized in the airway model to address the distinct effects of the triangular-shaped glottal aperture on the generation, development, and decay of turbulence. One of the salient turbulence characteristics for the triangular aperture case was found to be the relatively high turbulence levels around the center region (2r/D approximately 0) in conjunction with the asymmetric mean axial velocity across the frontal-rear (A-O-P) plane of the trachea at one tracheal diameter (x/D = 1) downstream from the glottis. The detailed turbulence properties such as the Reynolds shear stresses and turbulence intensities for the triangular aperture case differed significantly from those for the circular aperture case within a few tracheal diameters (x/D < 7) downstream from the apertures. The glottis-induced turbulence was incipient during the acceleration phase of inspiration and convected downstream with the traits of decaying turbulence.     

Purification, amino acid composition and N-terminal sequence of the major protein (protein H) of the outer membrane of Pasteurella multocida]

The major protein (protein H) of the outer membrane of Pasteurella multocida was purified by size-exclusion chromatography after selective extraction with detergents. The protein forms homotrimers which are stable in the presence of SDS at room temperature. Upon treatment at 100 degrees C, the protein is fully dissociated by the detergent into monomers exhibiting an apparent molecular mass of 37 kDa as estimated by electrophoresis. The amino acid composition of protein H is characterized by a low hydropathy index (HI = -0.40) and is strongly related to the compositions of bacterial porins, notably porins P2 (Haemophilus influenzae), PIA (Neisseria gonorrhoeae) and Cl.2 ("class 2 porin" of N. meningitidis). The N-terminal amino acid sequence of protein H shares a strong homology with those of porins OmpC (Escherichia coli) and P2. These data indicate that protein H of P. multocida is a porin belonging to the superfamily of the non-specific porins of Gram-negative eubacteria outer membrane.     

Regulation of juvenile hormone esterase gene expression in the tobacco budworm (Heliothis virescens)

The tissue distribution, developmental control, and induction of juvenile hormone esterase (JHE) mRNA was examined in Heliothis virescens using an 800-base pair fragment of a JHE cDNA clone. Northern hybridization analysis of poly(A)+RNA from fat body and integument of fifth stadium larvae indicated the presence of a single JHE mRNA species having an estimated length of 3 kilobases. On Day 2 of the fifth stadium (L5D2), basal JHE mRNA levels were 3-fold higher in the integument than the fat body, which correlated with the higher specific activity of the enzyme in the integument at this time. However, JHE mRNA levels in the fat body on Day 4 of the fifth stadium were 9-fold higher than on Day 2, while mRNA levels in the integument remained the same. This endogenous increase in JHE mRNA and activity in the fat body occurred at the time of peak hemolymph JHE activity. JHE mRNA was not detected in third stadium larvae which have very low levels of JHE activity. Treatment of L5D2 larvae with the juvenile hormone mimic epofenonane resulted in a 7- and 14-fold increase in the level of JHE mRNA in the integument and fat body, respectively. The mRNA induced in both tissues was of the same estimated length as the constitutively expressed message. The data indicate that the developmental regulation and induction of JHE can occur at the level of mRNA. There is evidence that the fat body secretes more JHE than does the integument and could be the major source of hemolymph JHE.