Phase High Pressure Liquid Chromatography Research Articles

Identification of the Substrate-binding Sites of 2′-5′-Oligoadenylate Synthetase

2'-5'-Oligoadenylate synthetases are interferon-induced enzymes that upon activation by double-stranded RNA polymerize ATP to 2'-5'-linked oligoadenylates. In our continuing effort to understand the mechanism of catalysis by these enzymes, we used photo affinity cross-linking and peptide mapping to identify the substrate-binding sites of the P69 isozyme of human 2'-5'-oligoadenylate synthetases. Radiolabeled azido 2'-5'-oligoadenylate dimers were enzymatically synthesized and used as ligands for cross-linking to the P69 protein by exposure to ultraviolet light. The radiolabeled protein was digested with trypsin, and two ligand-cross-linked peptides were purified by immobilized aluminum affinity chromatography followed by reverse phase high pressure liquid chromatography. The peptides were identified by mass spectrometry and peptide sequencing and were found to correspond to residues 420-425 and 539-547 of P69. To examine the functional importance of the cross-linking sites, specific residues in the two peptides were mutated. When residues in the two sites were mutated individually, ligand cross-linking was selectively eliminated at the mutated site, and the enzyme activity was lost almost completely. Using substrates that can serve either as a donor or as an acceptor but not both, we could identify one of the sites as the acceptor and the other as the donor site.

Comparative study on characteristics of lysozymes from the hemolymph of three lepidopteran larvae, Galleria mellonella, Bombyx mori, Agrius convolvuli

Lysozymes were purified from the hemolymph of three immunized Lepidopteran larvae, Galleria mellonella, Bombyx mori, Agrius convolvuli to compare their physico-chemical properties and antibacterial activities with those of chicken lysozyme. Four lysozymes including the one from chicken had similar molecular masses and chromatographic behavior on reverse phase-high pressure liquid chromatography. Western blotting analysis using an antibody raised against G. mellonella revealed that lysozyme cross-reacted with two other insect lysozymes but not with commercial chicken lysozyme. Antibacterial activities of lysozymes were measured in two types of tests: radial diffusion assay and colony count assay. Our antibacterial tests revealed that all lysozymes have strong activities against Gram-positive bacteria and three insect lysozymes still retain a little potency against Gram-negative bacteria, while chicken lysozyme has no activity against Gram-negative bacteria. Taken together, we conclude three Lepidopteran lysozymes have a common distinct structure and have an antibacterial activity, which is absent in chicken lysozyme, against Gram-negative bacteria.

Role of contaminating platelets in thromboxane synthesis in primary cultures of human umbilical vein endothelial cells

Previous studies suggested that cultured human endothelial cells metabolize arachidonic acid to thromboxane A 2. When primary cultures of human umbilical vein endothelial cells were incubated with 14 C -arachidonic acid and the 14 C -metabolites resolved by reverse phase high pressure liquid chromatography, radioactive products were observed that comigrated with 6-keto-prostaglandin F 1α and thromboxane B 2, the degradation products of prostacyclin and thromboxane A 2, respectively. Since platelets synthesize thromboxane A 2, the present study examined the hypothesis that adherent platelets may contaminate the primary cultures of human umbilical vein endothelial cells and be responsible for thromboxane B 2 production. Confluent primary cultures or passaged cells were stimulated with histamine (10 −5 M). Incubation buffer was analyzed by specific radioimmunoassays for 6-keto-prostaglandin F 1α and thromboxane B 2. The production of thromboxane B 2 decreased in the passaged cells (207±44 pg/ml versus 65±12 pg/ml; primary versus passaged cells). A moderate decrease in the yield of 6-keto-prostaglandin F 1α was measured in the passaged cells compared to the primary cultures (3159±356 pg/ml versus 1678±224 pg/ml, primary versus passaged cells). If the primary cultures were incubated with human platelet-rich plasma for 30 min prior to stimulation with histamine, the amount of thromboxane B 2 increased approximately 10-fold. In an additional experiment, sub-confluent primary cells were incubated with platelet-rich plasma for 30 min, washed to remove non-adherent platelets, and allowed to reach confluency. Confluent cells were then passaged and stimulated with histamine. The amount of thromboxane B 2 was not significantly different from that obtained with passaged cells that had not been incubated with platelet-rich plasma during the primary culture (83±15 pg/ml versus 65±12 pg/ml, respectively). If the cyclooxygenase inhibitor indomethacin was included in the incubations, the amounts of both thromboxane B 2 and 6-keto-prostaglandin F 1α decreased. In contrast, the thromboxane A 2 synthase inhibitor dazoxiben blocked thromboxane production and had no effect on the amount of 6-keto-prostaglandin F 1α. Light microscopy revealed the presence of adherent platelets in primary cultures with and without platelet-rich plasma but no platelets were observed in any group of passaged cells. Histofluorescence for platelet serotonin indicated the presence of platelets only in primary cultures of human umbilical vein endothelial cells or in cultures pre-incubated with platelet-rich plasma. These studies suggest that primary cultures of human umbilical vein endothelial cells contain adherent platelets that contribute to thromboxane synthesis.

Preparation of stable insulin-loaded nanospheres of poly(ethylene glycol) macromers and N-isopropyl acrylamide

A series of nanospheres composed of temperature-sensitive poly(N-isopropylacrylamide), poly(ethylene glycol) 400 dimethacrylate, and poly(ethylene glycol) 1000 methacrylate was prepared by a thermally-initiated free radical dispersion polymerization method. Insulin was loaded into the nanoparticles by equilibrium partitioning. The loading capacity of insulin into the nanoparticles was 2.1% (2.1 mg insulin/100 mg nanoparticles). The stability of the loaded insulin at elevated temperatures was investigated by reverse phase high pressure liquid chromatography. The nanoparticles were able to protect the loaded insulin, as more than 80% of the loaded insulin could still be detected compared to 0% for the control (0.1% insulin solution in PBS) when heated to 80°C for 5 h. The stability of the loaded insulin at high shear stress (289 1/s) was also investigated. No significant loss of insulin was detected both from nanoparticles loaded with insulin sample and the control (0.1% insulin solution in PBS). The results showed that shear stress alone did not have a major effect on insulin denaturation. The ability of the nanoparticles to protect the insulin from high temperature and high shear stress made the system a good candidate as a carrier for insulin for fluidized bed coating technology.

The Folding Pathway of α-Lactalbumin Elucidated by the Technique of Disulfide Scrambling: ISOLATION OF ON-PATHWAY AND OFF-PATHWAY INTERMEDIATES

The technique of disulfide scrambling permits reversible conversion of the native and denatured (scrambled) proteins via shuffling and reshuffling of disulfide bonds. Under strong denaturing conditions (e.g. 6 m guanidinium chloride) and in the presence of a thiol initiator, alpha-lactalbumin (alphaLA) denatures by shuffling its four native disulfide bonds and converts to an assembly of 45 species of scrambled isomers. Among them, two predominant isomers, designated as X-alphaLA-a and X-alphaLA-d, account for about 50% of the total denatured structure of alphaLA. X-alphaLA-a and X-alphaLA-d, which adopt the disulfide patterns of (1-2,3-4,5-6,7-8) and (1-2,3-6,4-5,7-8), respectively, represent the most unfolded structures among the 104 possible scrambled isomers (Chang, J.-Y., and Li, L. (2001) J. Biol. Chem. 276, 9705-9712). In this study, X-alphaLA-a and X-alphaLA-d were purified and allowed to refold through disulfide scrambling to form the native alphaLA. Folding intermediates were trapped kinetically by acid quenching and analyzed quantitatively by reversed phase high pressure liquid chromatography. The results revealed two major on-pathway productive intermediates, two major off-pathway kinetic traps, and at least 30 additional minor transient intermediates. Of the two major on-pathway intermediates, one takes on a native-like alpha-helical domain, and the other comprises a structured beta-sheet, calcium binding domain. The two major kinetic traps are apparently stabilized by locally formed non-native-like structures. Overall, the folding mechanism of alphaLA is essentially congruent with the model of "folding funnel" furnished with a rather intricate energy landscape.

Identification of biodegradation products formed by L-phenylalanine based segmented polyurethaneureas

The degradation of novel biodegradable segmented polyurethanes was investigated with a view to determining the cleavage points within the polymer backbones targeted by the enzyme chymotrypsin. While the materials were developed with specific enzyme cleavage sites designed into the polymer chains, the nature of their degradation had not yet been determined. In this work, two segmented polyurethaneureas containing L-phenylalanine residues in the chain extender and two control polymers were subjected to degradation in the presence of chymotrypsin. Samples were collected for analysis over a time period from 1 day to 8 weeks. The degradation products from these materials were isolated using solid phase extraction and reversed phase high pressure liquid chromatography, and identified using mass and tandem mass spectrometry. Three hard segment related degradation products were identified and provide important insight into the polyurethane backbone cleavage sites. Cleavage of urea, ester and urethane bonds were observed. The results confirmed that chymotrypsin was able to cleave ester bonds adjacent to phenylalanine residues contained within the novel chain extender.

Diethyl Phthalate, a Chemotactic Factor Secreted by Helicobacter pylori

The structure of a small-molecule, non-peptide chemotactic factor has been determined from activity purified to apparent homogeneity from Helicobacter pylori supernatants. H. pylori was grown in brucella broth media until one liter of solution had 0.9 absorbance units. The culture was centrifuged, and the bacteria re-suspended in physiological saline and incubated at 37 degrees C for 4 h. A monocyte migration bioassay revealed the presence of a single active chemotactic factor in the supernatant from this incubation. The chemotactic factor was concentrated by solid phase chromatography and purified by reverse phase high pressure liquid chromatography. The factor was shown to be indistinguishable from diethyl phthalate (DEP) on the basis of multiple criteria including nuclear magnetic resonance spectroscopy, electron impact mass spectroscopy, UV visible absorption spectrometry, GC and high pressure liquid chromatography retention times, and chemotactic activity toward monocytes. Control experiments with incubated culture media without detectable bacteria did not yield detectable DEP, suggesting it is bacterially derived. It is not known if the bacteria produce diethyl phthalate de novo or if it is a metabolic product of a precursor molecule present in culture media. DEP produced by H. pylori in addition to DEP present in man-made products may contribute to the high levels of DEP metabolites observed in human urine. DEP represents a new class of chemotactic factor.

The determination of ferulic acid in sugar beet pulp

The content of ferulic acid in sugar beet pulp was determined by reverse phase high-pressure liquid chromatography (HPLC) and UV/VIS-spectroscopy. The acid extracts of pectin carrying feruloyl groups were prepared for analysis. To release ferulic acid from pectin the hydrolysis in alkaline medium (pH = 12.5) was performed. Both non-hydrolysed and hydrolysed extracts were measured by UV/VIS-spectroscopy after pH adjustment to the value of 10. The absorbance maximum was observed at 372 nm (ester of ferulic acid) for non-hydrolysed extracts and at 345 nm (sodium ferulate) for hydrolysed extracts. The HPLC estimation of ferulic acid was made in hydrolysed extracts only. The content of ferulic acid in sugar beet pulp was in the range of 0.3–0.9% (m/m). The data obtained by application of the particular methods to one set of samples were statistically compared. The results of all methods were in good agreement with each other\

Phospholipase A2–derived neutral lipids from posthemorrhagic shock mesenteric lymph prime the neutrophil oxidative burst

Background. Our previous work identified posthemorrhagic shock mesenteric lymph (PHSML) lipids as key elements in polymorphonuclear neutrophil (PMN)–provoked acute lung injury. We hypothesize that gut phospholipase A2 (PLA2) is responsible for the generation of proinflammatory lipids in PHSML that primes circulating PMNs for enhanced oxidative burst. Methods. Mesenteric lymph was collected from rats (n = 5) before (preshock), during the induction of hemorrhagic shock (mean arterial pressure, 40 mm Hg × 30 minutes), and at resuscitation (shed blood + 2× lactated Ringer's solution). PLA2 inhibition (quinacrine, 10 mg/kg, intravenously) was given before shock was induced. Extracted lipids were separated by normal phase high-pressure liquid chromatography and resuspended in albumin. PMNs were exposed to a 5% vol:vol concentration of eluted lipids and activated with N-formyl-methionyl-leucyl-phenylalanine (1 μmol/L). Superoxide production was assessed by cytochrome C reduction. Results. High-pressure liquid chromatography–extracted neutral lipids of lymph collected before hemorrhagic shock did not prime the PMN oxidase, whereas isolated neutral lipids of postshock lymph primed PMNs 2.6- ± 0.32-fold above baseline (P <.05). PLA2 inhibition returned PHSML neutral lipid priming to baseline levels. Conclusions. PLA2 inhibition before hemorrhagic shock abrogates the neutrophil priming effects of PHSML through reduction of the accumulation of proinflammatory neutral lipids. Identification of these PLA2-dependent lipids provides a mechanistic link that may have therapeutic implications for postshock acute lung injury. (Surgery 2001;130:198-203.)

Identification of neuropeptide FF-related peptides in rodent spinal cord

Peptides which should be generated from the neuropeptide FF (NPFF) precursor were identified in mouse and rat spinal cord, by using reverse phase high pressure liquid chromatography with radioimmunoassay and electrospray mass spectrometry detection. In both species, two octapeptides, NPFF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) and NPSF (Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-amide) were identified but a longer peptide NPA-NPFF (Asn-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was present at the highest concentration in rat spinal cord. In mouse, the homologous peptide, SPA-NPFF (Ser-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was not detected. Both peptides NPFF and NPSF reverse morphine-induced analgesia in the tail flick test. Our data reveal species differences in the maturation of NPFF precursor.

Neem the source of versatile chemicals, Azadirachtin in modern pest management: a review

Neem is known for its therapeutic and pesticidal applications. Azadirachtin is the chemical constituent in neem seed kernel which is active against wide spectrum of insect pests. Beside azadirachtin several other chemical constituents such as salannin, meliantriol, etc., are also known to possess biocidal activity. The azadirachtin content in seed kernel generally varies between 0.1% to 0.7% and is determined using reverse phase high-pressure liquid chromatography (HPLC) system. For commercial application azadirachtin content 1500 ppm in extract-based and 300 ppm in oil-based products is an important requirement. Almost all parts of the neem tree are known for its medicinal value. Several neem compounds such as nimbin, nimbidin, nimbinin, etc., are found active for various medicinal applications.

Novel cytokinins: The predominant forms in mature buds of Pinus radiata.

To elicit the roles of cytokinins in the regulation of maturation of Pinus radiata D. Don, the spectrum of endogenous cytokinins and their concentration in the mature buds were analysed using double-solvent extraction, column complex purification and separation, a novel immunoaffinity purification method, normal and reverse phase high-pressure liquid chromatography, enzymatic treatment, radioimmunoassay and electrospray MS/MS spectrometry. We have isolated two novel cytokinin glycosides whose proposed structures are isopentenyladenine-9-(glucopyranosyl riboside), dihydrozeatin-9-(glucopyranosyl riboside) and confirmed the presence of zeatin-9-(glucopyranosyl riboside). We have also found the presence of novel phosphorylated forms of these 3 cytokinin ribosyl-linked glycosides. Quantitative analyses revealed that the cytokinin ribosyl-linked glycosides predominate in P. radiata mature buds. Although cytokinin free base, riboside and nucleotide forms are also present, we could find no evidence of the traditional cytokinin O- or N-glucosides in the conifer buds. Thus, cytokinin metabolism in mature buds of P. radiata is very different from other species previously examined.

Long-range (1)H-(15)N heteronuclear shift correlation at natural abundance: a tool to study benzothiazole biodegradation by two rhodococcus strains.

The biodegradation of benzothiazole and 2-hydroxybenzothiazole by two strains of Rhodococcus was monitored by reversed phase high-pressure liquid chromatography and by (1)H nuclear magnetic resonance (NMR). Both xenobiotics were biotransformed into a hydroxylated derivative of 2-hydroxybenzothiazole by these two strains. The chemical structure of this metabolite was determined by a new NMR methodology: long-range (1)H-(15)N heteronuclear shift correlation without any previous (15)N enrichment of the compound. This powerful NMR tool allowed us to assign the metabolite structure to 2,6-dihydroxybenzothiazole.

Tannic-membrane interactions on ultrafiltration of cork processing wastewaters

Cork processing wastewaters are very complex mixtures of vegetal extracts and have, among other natural compounds, a very high content of phenolic/tannic colloidal matter, that is responsible for severe problems of membrane fouling and drastic flux decline. In the present work the identification and quantification of phenolic/tannic compounds, responsible for membrane fouling, is carried out through the use of reverse phase high pressure liquid chromatography (HPLC). The compounds studied were gallic acid, protocatechuic acid, vanillic acid, syringic acid, ferulic acid and ellagic acid. The very different characteristics of these compounds in terms of hydrophilicity/hydrophobicity were correlated to their rejection coefficients for two cellulose acetate ultrafiltration membranes of 6000- and 98000-Da molecular weight cut-off (MWCO). The ultrafiltration transmembrane pressures ranged from 0.5 to 4 bar and the feed flowrate from 100 to 200 l/h. The more hydrophobic component (the ellagic acid) is almost totally retained and that independently of the membrane MWCO, transmembrane pressure, and feed flowrate leading to the conclusion of formation of an adsorption layer responsible for membrane fouling.

Biologically Active Recombinant Human Progastrin6–80Contains a Tightly Bound Calcium Ion

Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo. The aims of this study were to prepare recombinant human progastrin(6-80) and to investigate its structure and biological activities in vitro. Human progastrin(6-80) was expressed in Escherichia coli as a glutathione S-transferase fusion protein. After thrombin cleavage progastrin(6-80) was purified by reverse phase high pressure liquid chromatography and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry. Assays for metal ions by atomic emission spectroscopy revealed the presence of a single tightly bound calcium ion. Progastrin(6-80) at concentrations in the pm to nm range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC. The observations that progastrin(6-80) did not bind to either the cholecystokinin (CCK)-A or the gastrin/CCK-B receptor expressed in COS cells and that antagonists selective for either receptor did not reverse the proliferative effects of progastrin(6-80) suggested that progastrin(6-80) stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor. We conclude that recombinant human progastrin(6-80) is biologically active and contains a single calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin, this is the first report of selective, high affinity binding of metal ions to a prohormone.

Proteolytic Cleavage of Chromogranin A (CgA) by Plasmin: SELECTIVE LIBERATION OF A SPECIFIC BIOACTIVE CgA FRAGMENT THAT REGULATES CATECHOLAMINE RELEASE

Chromogranin A (CgA), the major soluble protein in catecholamine storage vesicles, serves as a prohormone that is cleaved into bioactive peptides that inhibit catecholamine release, providing an autocrine, negative feedback mechanism for regulating catecholamine responses during stress. However, the proteases responsible for the processing of CgA and release of bioactive peptides have not been established. Recently, we found that chromaffin cells express components of the plasmin(ogen) system, including tissue plasminogen activator, which is targeted to catecholamine storage vesicles and released with CgA and catecholamines in response to sympathoadrenal stimulation, and high affinity cell surface receptors for plasminogen, to promote plasminogen activation at the cell surface. In the present study, we investigated processing of CgA by plasmin and sought to identify specific bioactive CgA peptides produced by plasmin proteolysis. Highly purified human CgA (hCgA) was produced by expression in Escherichia coli and purification using metal affinity chromatography. hCgA was digested with plasmin. Matrix-assisted laser desorption/ionization mass spectrometry identified a major peptide produced with a mass/charge ratio (m/z) of 1546, corresponding uniquely to hCgA-(360-373), the identity of which was confirmed by reverse phase high pressure liquid chromatography and amino-terminal microsequencing. hCgA-(360-373) was selectively liberated by plasmin from hCgA at early time points and was stable even after prolonged exposure to plasmin. The corresponding synthetic peptide markedly inhibited nicotine-induced catecholamine release from pheochromocytoma cells. These results identify plasmin as a protease, present in the local environment of the chromaffin cell, that selectively cleaves CgA to generate a bioactive fragment, hCgA-(360-373), that inhibits nicotinic-mediated catecholamine release. These results suggest that the plasminogen/plasmin system through its interaction with CgA may play a major role in catecholaminergic function and suggest a specific mechanism as well as a discrete CgA peptide through which this effect is mediated.

CDNA Cloning and Functional Analysis of Ascidian Sperm Proacrosin

cDNA cloning and functional analysis of proacrosin from the ascidian Halocynthia roretzi were undertaken. The isolated cDNA of the ascidian preproacrosin consists of 2367 nucleotides, and an open reading frame encodes 505 amino acids, which corresponds to the molecular mass of 55,003 Da. The mRNA of proacrosin was found to be specifically expressed in the gonad by Northern blotting and in the spermatocytes or spermatids by in situ hybridization. The amino acid sequences around His(76), Asp(132), and Ser(227), which make up a catalytic triad, showed high homology to those of the trypsin family. Ascidian acrosin has paired basic residues (Lys(56)-His(57)) in the N-terminal region, which is one of the most characteristic features of mammalian acrosin. This region seems to play a key role in the binding of (pro)acrosin to the vitelline coat, because the peptide containing the paired basic residues, but not the peptide substituted with Ala, was capable of binding to the vitelline coat. Unlike mammalian proacrosin, ascidian proacrosin contains two CUB domains in the C-terminal region, in which CUB domain 1 seems to be involved in its binding to the vitelline coat. Four components of the vitelline coat that are capable of binding to CUB domain 1 in proacrosin were identified. In response to sperm activation, acrosin was released from sperm into the surrounding seawater, suggesting that ascidian acrosin plays a key role in sperm penetration through the coat. These results indicate that ascidian sperm contains a mammalian acrosin homologue, a multi-functional protein working in fertilization.

Human neutrophil-mediated nonoxidative antifungal activity against Cryptococcus neoformans.

It has long been appreciated that polymorphonuclear leukocytes (PMN) kill Cryptococcus neoformans, at least in part via generation of fungicidal oxidants. The aim of this study was to examine the contribution of nonoxidative mechanisms to the inhibition and killing of C. neoformans. Treatment of human PMN with inhibitors and scavengers of respiratory burst oxidants only partially reversed anticryptococcal activity, suggesting that both oxidative and nonoxidative mechanisms were operative. To define the mediators of nonoxidative anticryptococcal activity, PMN were fractionated into cytoplasmic, primary (azurophil) granule, and secondary (specific) granule fractions. Incubation of C. neoformans with these fractions for 18 h resulted in percent inhibition of growth of 67.4 +/- 3.4, 84.6 +/- 4.4, and 29.2 +/- 10.5 (mean +/- standard error, n = 3), respectively. Anticryptococcal activity of the cytoplasmic fraction was abrogated by zinc and depletion of calprotectin. Antifungal activity of the primary granules was significantly reduced by pronase treatment, boiling, high ionic strength, and magnesium but not calcium. Fractionation of the primary granules by reverse phase high-pressure liquid chromatography on a C(4) column over an acetonitrile gradient revealed multiple peaks with anticryptococcal activity. Of these, peaks 1 and 6 had substantial fungistatic and fungicidal activity. Peak 1 was identified by acid-urea polyacrylamide gel electrophoresis (PAGE) and mass spectroscopy as human neutrophil proteins (defensins) 1 to 3. Analysis of peak 6 by sodium dodecyl sulfate-PAGE revealed multiple bands. Thus, human PMN have nonoxidative anticryptococcal activity residing principally in their cytoplasmic and primary granule fractions. Calprotectin mediates the cytoplasmic activity, whereas multiple proteins, including defensins, are responsible for activity of the primary granules.

Selective Elimination of High Constitutive Activity or Chemokine Binding in the Human Herpesvirus 8 Encoded Seven Transmembrane Oncogene ORF74

Open reading frame 74 (ORF74) encoded by human herpesvirus 8 is a highly constitutively active seven transmembrane (7TM) receptor stimulated by angiogenic chemokines, e.g. growth-related oncogene-alpha, and inhibited by angiostatic chemokines e.g. interferon-gamma-inducible protein. Transgenic mice expressing ORF74 under control of the CD2 promoter develop highly vascularized Kaposi's sarcoma-like tumors. Through targeted mutagenesis we here create three distinct phenotypes of ORF74: a receptor with normal, high constitutive signaling through the phospholipase C pathway but deprived of binding and action of chemokines obtained through deletion of 22 amino acids from the N-terminal extension; an ORF74 with high constitutive activity but with selective elimination of stimulatory regulation by angiogenic chemokines obtained through substitution of basic residues at the extracellular ends of TM-V or TM-VI; and an ORF74 lacking constitutive activity but with preserved ability to be stimulated by agonist chemokines obtained through introduction of an Asp residue on the hydrophobic, presumed membrane-exposed face of TM-II. It is concluded that careful molecular dissection can selectively eliminate either agonist or inverse agonist modulation as well as high constitutive activity of the virally encoded oncogene ORF74 and that these mutant forms presumably can be used in transgenic animals to identify the molecular mechanism of its transforming activity.

Isolation of MFO inducers from tissues of white suckers caged in bleached kraft mill effluent

White sucker ( Catostomus commersoni) caged for 3 days in a bleached kraft mill effluent (BKME) stream had elevated mixed function oxygenase (MFO) activities 15–90 fold those of fish caged in a reference stream. Liver composites of male and female fish were ground and extracted with dichloromethane (DCM), methanol or 50% DCM/methanol, and tested for MFO activity in rat hepatoma cells (H4IIE). There was no difference in the potency of H4IIE EROD induction among the three solvents, so DCM extracts were split into 31 fractions using reverse phase high pressure liquid chromatography (HPLC). H4IIE MFO activity was elevated in several fractions, with three early peaks and several later peaks of induction, indicating several classes or compounds causing MFO induction were present in the fish livers. Polychlorinated dibenzo- p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and chlorinated diphenylethers (CDPEs) were detected in several late-eluting fractions, but concentrations were not high enough for these compounds to be solely responsible for the observed induction. Induction by liver extracts decreased as cell exposure times increased (24, 48 or 72 h), suggesting that some inducers were more easily metabolized and eliminated from the H4IIE cells. In contrast, 2,3,7,8-tetrachlorodibenzo- p-dioxin (2378-TCDD) had similar potency over 24, 48 and 72 h, as it was relatively resistant to metabolism.