Joseph M. Kinkade

Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox

High molecular weight homologues of gp91phox, the superoxide-generating subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, have been identified in human (h) and Caenorhabditis elegans (Ce), and are termed Duox for “dual oxidase” because they have both a peroxidase homology domain and a gp91phox domain. A topology model predicts that the enzyme will utilize cytosolic NADPH to generate reactive oxygen, but the function of the ecto peroxidase domain was unknown. Ce-Duox1 is expressed in hypodermal cells underlying the cuticle of larval animals. To investigate function, RNA interference (RNAi) was carried out in C. elegans. RNAi animals showed complex phenotypes similar to those described previously in mutations in collagen biosynthesis that are known to affect the cuticle, an extracellular matrix. Electron micrographs showed gross abnormalities in the cuticle of RNAi animals. In cuticle, collagen and other proteins are cross-linked via di- and trityrosine linkages, and these linkages were absent in RNAi animals. The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed cross-linking of free tyrosine ethyl ester. Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix.

Isolation of three native forms of myeloperoxidase from human polymorphonuclear leukocytes

Myeloperoxidase @IPO) is an unusual and complex heme-containing glycoprotein [ 1,2] which plays an important functional role in the oxidative metabolism of PMN from many sources [3]. The isolation and purification of this enzyme [l] has been described and fluorochrome-labeled antibodies made against highly purified human MPO have been useful in studies aimed at elucidating the role of MPO-containing azurophilic granules in phagocytosis by PMN [4]. However, there appear to be multiple forms of MPO which have different properties [5-81. Polyacrylamide gel electrophoresis data in [5,7] support the idea that human MPO consists of a number of isoenzymes. Often, the genetic origin of the forms was not clear because PMN had been pooled from many donors. enzyme treatment [lo]. However, according to [ 1 l] many commonly used procedures for preventing proteolysis, such as use of PMSF and divalent cation chelators, are not effective in the isolation of intact proteins from human PMN; only the use of DFP prevented proteolysis [ 111. Here, we present evidence that MPO from a single human donor can be resolved into 3 distinct chromatographic species which are native to PMN, which have the same Mr-values, but which exhibit different sensitivities to inhibition by AT. This is the first description of a chromatographic procedure for the preparative and reproducible isolation of these different forms of MPO from normal, human PMN. Further studies on the biochemical and functional characterization of these enzymes should now be possible.

Identification of lactate dehydrogenase-M polypeptide translated in vitro from human and mouse tumor cell poly(A)-containing messenger RNA

Rabbit anti-human lactate dehydrogenase-5(M4) antisera were raised which cross-reacted with mouse lactate dehydrogenase M polypeptide. The antisera were used for identification of human and mouse LDH-M polypeptides synthesized using an in vitro system directed by the mRNAs. The in vitro translation products directed by both mRNAs were similar in size and immunologically identical to the authentic LDH-M polypeptides. The sizes of the mRNAs encoding for both human and mouse LDH-M polypeptides were similar, about 15S (1445 nucleotides) and were shorter than the corresponding rat mRNA which is about 18S (1765 nucleotides).

Colony Stimulating Factor (CSF) from Human Leukemic Urine: Affinity Chromatography and Isoelectric Focusing

Summary Biological activities associated with colony-stimulating factor (CSF) from human leukemic urine were found to be selectively retained on an affinity adsorbent of Con A-Sepharose. Elution of activity was achieved using a linear gradient of either α-methyl-D-mannopyranoside (αMM) or α-methyl-D-glucopyranoside (αMG), and resulted in significant increases in specific biological activity. Rechromatography of appropriate fractions indicated that retention of CSF activities was not artifactual. Pretreatment of the affinity matrix with αMM completely inhibited binding of CSF. Affinity chromatography of CSF on a Blue Dextran-Sepharose adsorbent was found to be an effective method for removing albumin, a major protein contaminant in urinary preparations. Treatment of CSF with neuraminidase had no effect on its in vitro activity; however, such treatment resulted in an increase in the isoelectric point of CSF from pH 3.5 to pH 4.7, as determined using both sucrose and polyacrylamide gel stabilized pH gradients. Relatively broad regions of biological activity were observed following isoelectric focusing of both neuraminidasetreated and untreated CSF, suggesting that activity was associated with a heterogeneous/polydisperse population of molecular species. The authors thank E. S. Mingioli, M. Parker, D. LaVia, and B. Jambois for expert technical assistance. We are indebted to Dr. J. Mendicino for informing us about the properties of Blue Dextran-Sepharose. We also thank Dr. E. R. Stanley, who, following completion of the present studies, brought to our attention an earlier workshop report of related work, and, indirectly, a paper by Iscove et al. (34), who showed that colony-stimulating activity was retained on an affinity matrix similar to ours.

Effects of 5-bromo-2′-deoxyuridine (BUdR) on in vitro colony formation in cultures of mouse bond marrow cells

Colony stimulating factors (CSF) appear to be glycoproteins [1] and have been prepared from many sources including serum and urine of both normal and leukemic individuals [2]. The biological activity of CSF is generally measured by its ability to promote the formation of granulocyte and/or mononuclear cell colonies in cultures of bone marrow cells suspended in either soft agar [3, 4] or methyl cellulose [5]. CSF has been shown to stimulate the in vitro incorporation of labeled precursors into DNA [6 -8 ] , but colony formation cannot be explained simply in terms of a clonal proliferative model [8-11 ]. Moreover, morphological observations and histochemical evidence have suggested that CSF promotes the in vitro differentiation and/or selection of granulocyte and mononuclear cell precursors in both normal and leukemic marrow cell populations [ 12-14]. Low concentrations of the thymidine analog BUdR inhibit the differentiation of specific cell types and prevent normal development in embryos [ 15-17] . It was of interest, therefore, to investigate what effect low concentrations of BUdR might have upon CSFdependent colony formation. In this study, evidence is presented that CSF-dependent in vitro colony formation in cultures of mouse bone marrow cells is significantly inhibited by as little as 10 -7 M BUdR, and that BUdR appears to exert its inhibitory effect at a relatively early stage of colony formation.