Endogenous nitric oxide decreases xanthine oxidase-mediated neutrophil adherence: role of P-selectin

Terada, Lance S., John E. Repine, Dale Piermattei, andBrooks M. Hybertson. Endogenous nitric oxide decreases xanthine oxidase-mediated neutrophil adherence: role of P-selectin.J. Appl. Physiol. 82(3): 913-917, 1997.The oxygen radical-producing enzyme xanthine oxidase (XO) canpromote neutrophil adherence to endothelium. Recognizing that a balanceoften exists in inflammatory processes, we sought to determine whetherXO initiates antiadherent pathways. We found that bovine pulmonaryarterial endothelial cells (EC) exposed to XO released increasedamounts of nitrite into the media, reflecting an increased productionof nitric oxide (NO). When EC were subjected to shear stress, treatmentwith XO and/or the NO synthase inhibitorN-nitro-L-arginine(L-NNA) increased neutrophilrolling behavior and firm neutrophil adherence to EC in an additivefashion. Both rolling and adherent interactions were abolished bymonoclonal antibodies directed against P-selectin. In addition,treatment of EC with XO and/orL-NNA increased both surfaceexpression of P-selectin and release of von Willebrand factor intomedia. Finally, treatment of EC with the NO donor sodium nitroprussidedecreased XO-mediated neutrophil rolling and adherence. We concludethat XO stimulates EC to produce NO and that NO decreases theP-selectin-dependent neutrophil adhesion initiated by XO. Suchincreases in endogenous NO may constitute an importantnegative-feedback response to the acute proadhesive effects of XO.

     

Genetic and functional evidence that Type IV pili are required for social gliding motility in Myxococcus xanthus

The social gliding behaviour of Myxococcus xanthus has previously been associated with the presence of polar pili. A Tn 5 transposon insertion was isolated which introduces a defect in social gliding and is genetically linked to a known sgl locus; this insertion was found also to cause a piliation defect. A 2.7 kb section of DNA was isolated from either side of this transposon and sequenced, revealing three genes which encode amino acid sequences with substantial similarity to components of the Type IV pilus biogenesis pathway in Pseudomonas aeruginosa . The myxococcal pilA gene encodes a putative pilin precursor with a short signal sequence and processing site similar to those of other Type IV pilins. Myxococcal pilS and pilR encode amino acid sequences with similarity to PilS and PilR of P. aeruginosa , as well as to other members of the NtrB/C family of two-component regulators. Mutations within pilR and pilA that have no polar effect were demonstrated to be responsible for pilus and social motility defects. These results indicate that the pili of M. xanthus belong to the Type IV family of pili, and demonstrate that these pili are actually required for social motility.     

Lipoxygenase- and cyclooxygenase-reaction products and incorporation into glycerolipids of radiolabeled arachidonic acid in the bovine retina

The metabolism of radiolabeled arachidonic acid (AA) by the intact bovine retina has been studied. Synthesis of prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs), and incorporation of AA into glycerolipids has been measured by reverse-phase and straight-phase high performance liquid chromatography with flow scintillation detection, and by thin-layer chromatography. AA was actively acylated into glycerolipids, particularly triglycerides, phosphatidylcholine and phosphatidylinositol. AA was also converted to the major PGs, PGF_2α, PGE₂, PGD₂, 6-keto-PGF_1α and TXB₂, and to the lipoxygenase reaction products, 12-HETE, 5-HETE, and other monohydroxy isomers. Approximately 6% of the radiolabeled AA was converted to eicosanoids. The synthesis of HETEs was inhibited in a concentration-dependent manner (IC₅₀ = 8.3 NM) by nordihydroguaiaretic acid (NDGA). PG synthesis was inhibited by aspirin (10 μM), indomethacin (1 μM) and NDGA (IC₅₀ = 380 nM). Metabolism of AA via lipoxygenase, cyclooxygenase and activation-acylation was inhibited by boiling retinal tissue prior to incubation. These studies demonstrate an active system for the uptake and utilization of AA in the bovine retina, and provide the first evidence of lipoxygenase-mediated metabolism of AA, resulting in the synthesis of mono-hydroxyeicosatetraenoic acids, in the retina.     

Individual recognition in salamanders: Cloacal odours

Male red-backed salamanders (Plethodon cinereus) were studied to determine the origin of the chemical cues allowing for individual odour recognition between conspecifics. Individual males showed a preference for substrates with their own faecal and cloacal odours rather than those marked by unfamiliar conspecific males. This discriminatory ability did not depend on recent differences in food consumed. These scent marks might be used to define breeding territories in the field.     

A mutant of CHO-K1 cells deficient in two nonsequential steps of de novo purine biosynthesis

An unusual new purine-requiring mutant, Ade^?P_AB, of Chinese hamster ovary cells (CHO-K1) is described. Ade^?P_AB will grow in medium supplemented with hypoxanthine, adenine, or aminoimidazole carboxamide. Ade^?P_AB fails to show genetic complementation with either Ade^?A, defective in amidophosphoribosyltransferase (E.C. 2.4.2.14), or Ade^?B, defective in phosphoribosylformylglycinamidine FGAM) synthetase (E.C. 6.3.5.3.), but will complement all five of our other hypoxanthine-requiring Ade^? complementation groups. Analysis of purine synthesis in wild-type, mutant, and revertant cells and analysis of relevant enzyme activities in cell-free extracts prepared from these cells demonstrates that Ade^?P_AB is similar to Ade^?B in that it has lost FGAM synthetase activity, and is similar to Ade^?A in that it has lost glutamine-dependent amidophosphoribosyltransferase activity. Unlike Ade^?A, however, Ade^?P_AB retains the ability to synthesize phosphoribosylamine (PRA), the product of the amidophosphoribosyltransferase reaction, if NH₄Cl is substituted for glutamine as the nitrogen donor. Moreover, partial revertants of Ade^?P_AB can apparently synthesize sufficient purines for growth using the NH₄Cl-dependent reaction. The available evidence indicates that neither a double mutation nor a deletion is probable in Ade^?P_AB. We discuss the relevance of these observations for our understanding of both the regulation of purine biosynthesis in mammalian cells and the structural organization of the enzymes defective in Ade^?P_AB and the genes coding for these enzymes.     

Architecture of normal pancreas as revealed by retrograde injection

Summary The architecture of the pancreas was revealed by retrograde injection of the pancreatic ductal system of normal rats with a silicone rubber compound, and subsequent study of the preparation by light microscopy and scanning electron microscopy. The injected material became associated with both ducts and acinar areas. Examination of these specimens suggests that the arrangement of the exocrine pancreas is that of a complexly curving and branching system of tubules which anastomose and end blindly. This architecture, which is not that of a true acinar gland, provides a rational basis for the understanding of the simple dedifferentiative changes that accompany pancreatic carcinogenesis, and which have been generally interpreted as representing ductular proliferation.Supported by Grant Number CA22063, awarded by the National Cancer Institute, DHEW, through the National Pancreatic Cancer ProjectI thank Mr. Jackson Rhoads and Dr. J. Michael Barrett for information on preparative procedures     

Planning an adaptive numerical classification

Summary While numerical methods of classification seek to strictly formalise one part of the classificatory process, a human classifier uses a variety of different approaches to obtain a satisfactory organisation of data. In this paper some means of increasing the flexibility of numerical methods are discussed, in particular the possible uses of the taxonomic hierarchy. It is first necessary to show that higher taxonomic units contain ecologically interesting information. Then, using a two parameter classification method a means of progressing from higher to lower taxonomic units, during analysis and in a data dependent manner, is outlined. A similar procedure is possible with nested samples. However, since the value of a classification resides in its ecological interpretation, an automatic classification requires some means of ascribing meanings to classes. While such a semantic analysis seems theoretically possible, its practical attainment presents considerable difficulties.Contribution to the Symposium on Plant species and plant communities, held at Nijmegen, 11–12 November 1976, on the occasion of the 60th birthday of Professor Victor Westhoff.     

Purification and properties of soybean nodule xanthine dehydrogenase

Xanthine dehydrogenase (EC 1.2.1.37), an essential enzyme for ureide metabolism was purified from the cytosol fraction of soybean nodules. The purified xanthine dehydrogenase was shown to be homogeneous by electrophoresis and a pI of 4.7 was determined by isoelectric focusing. The enzyme had a molecular weight of 285,000 and two subunits of molecular weight 141,000 each. The holoenzyme contained 1.7 (±0.7) mol Mo and 8.1 (±2.0) mol Fe/mol enzyme and the enzyme also contained FMN and is thus a molybdoironflavoprotein. Soybean xanthine dehydrogenase is the second enzyme in plants demonstrated to contain Mo and the first xanthine-oxidizing enzyme reported to contain FMN, rather than FAD as the flavin cofactor.     

Vitreous Carbon: A New Material for Making Microtome Knives

The quality of sections obtained by microtomy depends to a large extent on the quality and characteristics of the microtome knife itself. Despite the need for improved microtomy techniques, there have been few significant developments since the introduction of glass and diamond knives in the 1950's. The manufacture of microtome knives from vitreous carbon provides new possibilities for developing both improved methods and improved equipment for specimen sectioning. Vitreous carbon has unique physical properties that lend themselves to the generation of precision cutting edges. Such an edge can be obtained either by breaking a piece of vitreous carbon or by using lapidary techniques. The resultant edge seems well adapted to both thick and thin sectioning. The introduction of vitreous carbon as a sectioning tool offers a significant alternative to metal, glass and diamond knives.