Kenneth B. Taylor

15-Lipoxygenase Catalytically Consumes Nitric Oxide and Impairs Activation of Guanylate Cyclase

Analysis of purified soybean and rabbit reticulocyte 15-lipoxygenase (15-LOX) and PA317 cells transfected with human 15-LOX revealed a rapid rate of linoleate-dependent nitric oxide (⋅NO) uptake that coincided with reversible inhibition of product ((13S)-hydroperoxyoctadecadienoic acid, or (13S)-HPODE) formation. No reaction of ⋅NO (up to 2 μm) with either native (Ered) or ferric LOXs (0.2 μm) metal centers to form nitrosyl complexes occurred at these ⋅NO concentrations. During HPODE-dependent activation of 15-LOX, there was consumption of 2 mol of ⋅NO/mol of 15-LOX. Stopped flow fluorescence spectroscopy showed that ⋅NO (2.2 μm) did not alter the rate or extent of (13S)-HPODE-induced tryptophan fluorescence quenching associated with 15-LOX activation. Additionally,⋅NO does not inhibit the anaerobic peroxidase activity of 15-LOX, inferring that the inhibitory actions of ⋅NO are due to reaction with the enzyme-bound lipid peroxyl radical, rather than impairment of (13S)-HPODE-dependent enzyme activation. From this, a mechanism of 15-LOX inhibition by ⋅NO is proposed whereby reaction of ⋅NO with EredLOO⋅ generates Ered and LOONO, which hydrolyzes to (13S)-HPODE and nitrite (NO 2 − ). Reactivation of Ered, considerably slower than dioxygenase activity, is then required to complete the catalytic cycle and leads to a net inhibition of rates of (13S)-HPODE formation. This reaction of ⋅NO with 15-LOX inhibited ⋅NO-dependent activation of soluble guanylate cyclase and consequent cGMP production. Since accelerated ⋅NO production, enhanced 15-LOX gene expression, and 15-LOX product formation occurs in diverse inflammatory conditions, these observations indicate that reactions of ⋅NO with lipoxygenase peroxyl radical intermediates will result in modulation of both ⋅NO bioavailability and rates of production of lipid signaling mediators.

Expression of a foreign gene in Chlamydomonas reinhardtii

Genomic transformation of Chlamydomonas reinhardtii exposed to glass-bead abrasion was accomplished with a chimeric neomycin phosphotransferaseII (NPTII)-encoding gene (nos::npt) flanked by the nopaline synthase promoter and polyadenylation sequences obtained from the Ti plasmid of Agrobacterium tumefaciens. These sequences were in a plasmid (pGA482) which also contained gene nit1 encoding nitrate reductase of C. reinhardtii. Transformants were selected by their ability to grow on medium containing nitrate, and 52% of these was also resistant to kanamycin. Evidence for nos::npt expression includes: (1) hybridization with probes specific for npt, (2) demonstration of NPTII activity after electrophoresis of extracts, and (3) chromatographic identification of the reaction product of NPTII, kanamycin phosphate. The highly biased codon usage in Chlamydomonas does not preclude expression.

Cyanobacteria carrying an smt-lux transcriptional fusion as biosensors for the detection of heavy metal cations

The metal-responsivesmt operator/promoter region ofSynechococcus PCC7942 was fused to theluxCDABE genes ofVibrio fischeri. Plasmid DNA (pJLE23) carrying this fusion conferred metal ion-inducible luminescence to transformed cyanobacteria.Synechococcus PCC7942 (pJLE23) was sensitive to ZnCl2 concentrations within a range of 0.5–4 μM as demonstrated by induction of luminescence. Trace levels of CuSO4, and CdCl2 were also detected.

Effect of the levels of dissolved oxygen on the expression of recombinant proteins in four recombinantEscherichia coli strains

SummaryFour recombinant strains ofEscherichia coli were examined for the effects of the dissolved oxygen level on the level of biomass, the plasmid content, and the level of recombinant protein at the stationary phase of batch growth. Strains JM101/pYEJ001, and TB-1/pYEJ001 (encoding chloramphenicol acetyltransferase), and strain TB-1/p1034, and TB-1/pUC19 (encoding β-galactosidase) were grown at the constant dissolved oxygen levels of 0, 50, and 100% air saturation, as well as in the absence of dissolved, oxygen control. The biomass of all strains under constant aerobic conditions was 12–36 times higher than that under anaerobic conditions, but was the same as or slightly higher than that without dissolved oxygen control. The plasmid content in all strains under anaerobic conditions was 2.9–11.7 times higher than that under aerobic conditions. The optimal dissolved oxygen concentration for the specific activity of recombinant proteins was dependent upon the strain. In no strain were constant aerobic conditions optimal. However, because of the effect on biomass, controlled aerobic conditions were optimal for the volumetric activity of recombinant protein in all but one strain.

The production of recombinant beta-galactosidase in Escherichia coli in yeast extract enriched medium

SummaryThe productivity ofEscherichia coli biomass and recombinant beta-galactosidase was increased in Luria broth (LB) enriched with yeast extract. In flask culture under conditions of LB limitation, yeast extract suplementation gave the highest biomass (strain HB101/pRW756) stimulation per unit of component added compared with supplementation by various amounts of amino acids, vitamins, minerals, purines/pyrimidines, tryptone, casamino acids, casein peptone or gelatin peptone. The biomass production ofE. coli HB101/pRW756, XL-1 blue/puc118, XL-1 Blue FF/puc118 and TB-1/p1034 cells was stimulated in fermentor-scale experiments with additional yeast extract in LB. Total beta-galactosidase production from plasmid genes in fermentor-scale experiments was increased 105.4% in XL-1 blue/puc118 cells, 365.5% in XL-1 blue FF/puc118 cells and 421.4% in TB-1/p1034 cells by 0.5%, 1% and 1% weight per volume of additional yeast extract in LB, respectively. Depending on different strains, the increase of the enzyme production was obtained either by increased biomass, or the combination of enhanced gene expression and increased biomass. Neither the biomass nor beta-galactosidase production was stimulated in N4830/p1034 cells by the increase in yeast extract concentration in the medium.

The fermentation of xylose: studies by carbon-13 nuclear magnetic resonance spectroscopy

SummaryThe fermentation ofd-xylose byPachysolen tannophilus, Candida shehatae, andPichia stipitis has been investigated by13C-nuclear magnetic resonance spectroscopy of both whole cells and extracts. The spectra of whole cells metabolizingd-xylose with natural isotopic abundance had significant resonance signals corresponding only to xylitol, ethanol and xylose. The spectra of whole cells in the presence of [1-13C]xylose or [2-13C]xylose had resonance signals corresponding to the C-1 or C-2, respectively, of xylose, the C-1 or C-2, respectively, of xylitol, and the C-2 or C-1, respectively, of ethanol. Xylitol was metabolized only in the presence of an electron acceptor (acetone) and the only identifiable product was ethanol. The fact that the amount of ethanol was insufficient to account for the xylitol metabolized indicates that an additional fate of xylitol carbon must exist, probably carbon dioxide. The rapid metabolism of xylulose to ethanol, xylitol and arabinitol indicates that xylulose is a true intermediate and that xylitol dehydrogenase catalyzes the reduction (or oxidation) with different stereochemical specificity from that which interconverts xylitol andd-xylulose. The amino acidl-alanine was identified by the resonance position of the C-3 carbon and by enzymatic analysis of incubation mixtures containing yeast and [1-13C]xylose or [1-13C]glucose. The position of the label from both substrates and the identification of isotope also in C-1 of alamine indicates flux through the transketolase/transaldolase pathway in the metabolism. The identification of a resonance signal corresponding to the C-1 of ethanol in spectra of yeast in the presence of [1-13C]xylose and fluoroacetate (but not arsenite) indicates the existence of equilibration of some precursor of ethanol (e.g. pyruvate) with a symmetric intermediate (e.g. fumarate or succinate) under these conditions.

Replacement of Conserved Cysteines in Human Tissue Inhibitor of Metalloproteinases-1

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is resistant to extremes of temperature and pH. This is thought to be due in part to the presence of six sulfhydryl bridges presumed to maintain the structural integrity of the molecule. As part of a study looking at structure-function relationships, a number of the conserved cysteine residues in TIMP-1 were targeted for replacement with serine. Single and double replacements of these conserved cysteines, as well as replacements around these cysteines, were expressed using a vaccinia virus system and analyzed for functional and structural competence. Analysis by circular dichroism indicated that these mutants maintained secondary structures similar to those of wild-type TIMP-1. Trypsin susceptibility experiments indicated that the tertiary structure of the mutants had not been drastically changed. Analysis of functional competence demonstrated that there were significant changes in some of these mutants. Assays using collagen fibrils or gelatin as substrates indicated that the double mutant C1S/C70S, but not C3S/C99S, had lost inhibitory activity against human fibroblast-type collagenase (FIB-CL) and at high concentrations only had slight activity againstM r 72,000 gelatinase (M r 72,000 gelatinase). Kinetic analysis of TIMP-1 inhibition of FIB-CL cleavage of a peptide substrate indicated that mutants C1S/C70S, C3S/C99S, and CEEC → CQQC retained their ability to inhibit FIB-CL in a manner similar to wild-type TIMP-1, while mutants C1S and C70S showed little inhibitory activity. The mutants C99S and C137S could also inhibit FIB-CL cleavage of the peptide substrate. The results indicated that the degree of inhibition by the TIMP-1 mutants varied somewhat depending on the choice of substrates. Interestingly, replacing both cysteines from a disulfide bond in the wild-type molecule resulted in a more competent inhibitor than either of the single site “parent” mutations. Taken together, these experiments indicate that TIMP-1 can be rendered inactive by the loss of a single cysteine.

Catalytic domain comparisons of human fibroblast-type collagenase, stromelysin-1, and matrilysin.

The propeptide plus the catalytic domain of human fibroblast-type collagenase, stromelysin-1, and matrilysin were expressed in Escherichia coli to directly compare the properties of all three catalytic domains utilizing the same assays. Truncated fibroblast-type collagenase (mini-CL), truncated stromelysin-1 (mini-SL-1), and matrilysin, like their native counterparts, could be activated by organomercurials, trypsin, or SDS. The mini-CL and mini-SL-1 displayed catalytic properties similar to their native counterparts, except that the mini-CL could not cleave native type I collagen. The k(cat)/Km for matrilysin (355 microM(-1) h(-1)) on the synthetic Mca-peptide was much higher than that for mini-CL (69 microM(-1) h(-1)) or mini-SL-1 (23.6 microM(-1) h(-1)). Mini-SL-1 and matrilysin, but not mini-CL, were capable of superactivating collagenase thus increasing the rate of collagen cleavage. Mini-CL and mini-SL-1, but not matrilysin, were able to form SDS-stable complexes with TIMP-1 when co-incubated with an organomercurial and TIMP-1. The second-order rate constant (k(on)) for TIMP-1 inhibition of mini-CL and mini-SL-1 were similar, 0.635 x 10(5) M(-1) s(-1) and 1.52 x 10(5) M(-1) s(-1), respectively. The k(on) for TIMP-1 inhibition of matrilysin was lower (0.130 x 10(5) M(-1) s(-1)) supporting the observation that no SDS stable complexes were detected. This study demonstrates that these catalytic domains are distinct and play a major role in the specificity of these enzymes in regard to rate of catalysis, TIMP-1 binding, and superactivation of collagenase.

Expression of mouse metallothionein in the cyanobacteriumSynechococcus PCC7942

A cDNA encoding mouse metallothionein was cloned into the shuttle vector pUC303, creating a translational fusion with the bacterial chloramphenicol acetyltransferase gene. The resulting fusion protein has been expressed in the cyanobacteriumSynechococcus PCC7942. Cyanobacterial transformants expressed mouse metallothionein-specific mRNA species as detected by RNA slot blots. In addition, the transformants expressed a unique cadmium ionbinding protein corresponding to the predicted size of the mouse metallothionein fusion protein. Expression of this fusion protein conferred a two-to five-fold increase in cadmium ion tolerance and accumulation onSynechococcus PCC7942.

Sanfilippo Syndrome, Type D: A Spectrophotometric Assay with Prenatal Diagnostic Potential

ABSTRACT: Sanfilippo syndrome, type D (MPS IIID), is characterized by moderate physical abnormalities, progressive mental deterioration, and deficient activity of N-acetylglucosamine 6-sulfate sulfatase, a lysosomal hydrolase involved in the degradation of heparin, keratan sulfate, and heparan sulfate. To date, demonstration of the enzyme deficiency typically relies on a radiolabeled trisaccharide substrate derived from heparan sulfate. In our study, we have developed a spectrophotometric assay for the determination of N-acetylglucosamine 6-sulfate sulfatase activity using the monosaccharide, N-acetylglucosamine 6-sulfate, as substrate. The reaction mixture was incubated for 6 h at 37°C and, after Dowex chromatography, released N-acetylglucosamine was measured by a modification of the method of Reissig. Assay conditions were optimized for cultured skin fibroblasts and primary cultures of amniotic fluid cells. The pH optimum for each was 5.5. The assay was linear for 24 h and up to 0.1 absorbance units. Activities of the three known MPS IIID skin fibroblast cell lines were more than 4 SD below the skin fibroblast control mean and more than 5 SD below the control mean for amniotic fluid cells. An enzyme deficiency in cultured amniotic fluid cells of the same magnitude as the skin fibroblasts of the known patients would be detectable and, therefore, prenatal diagnosis by this method is feasible.