Purification and characterization of a colony stimulating factor from human lung.

Conditioned medium prepared from human autopsy lung tissue contains high level activity of colony stimulating factor which stimulates granulocytes and macrophage colony formation in both mouse and human bone marrow. The lung colony stimulating factor has been purified about 2250-fold by methods including hydroxylapatite chromatography, preparative gel electrophoresis, preparative isoelectric focusing, and gel filtration chromatography. The final specific activity was 2.7 X 10(6) units/mg. The purified factor has a molecular weight of 41 000 as determined by gel filtration. It is stable at the pH range of 6.5--10 and 56 degrees C for 30 min but sensitive to protease digestion and periodate oxidation. On polyacrylamide gel electrophoresis, it migrates in the alpha-globulin post-albumin region. Upon isoelectrofocusing lung colony stimulating factor appears heterogeneous with isoelectric points of 3.7--4.3. Treatment with neuraminidase did not affect its activity, but caused a change in electrophoretic mobility and isoelectric point. Antibody produced by immunizing rabbits with partially purified lung colony stimulating factor exerted strong inhibitory activity on the factor from lung as well as on colony stimulating factor from other human sources including serum, urine, and placenta.     

Inheritance of leaf rust and stem rust resistance in 'Roblin' wheat.

The Canadian common wheat (Triticum aestivum L.) cultivar 'Roblin' is resistant to both leaf rust (Puccinia recondita Rob. ex. Desm.) and stem rust (Puccinia graminis Pers. f. sp. tritici Eriks. and E. Henn.). To study the genetics of this resistance, 'Roblin' was crossed with 'Thatcher', a leaf rust susceptible cultivar, and RL6071, a stem rust susceptible line. A set of F6 random lines was developed from each cross. The random lines and the parents were grown in a field rust nursery artificially inoculated with a mixture of P. recondita and P. graminis isolates and scored for rust reaction. The same material was tested with specific races of leaf rust and stem rust. These data indicated that 'Roblin' has Lr1, Lr10, Lr13, and Lr34 for resistance to P. recondita and Sr5, Sr9b, Sr11, and possibly Sr7a and Sr12 for resistance to P. graminis. In a 'Thatcher' background, the presence of Lr34 contributes to improve stem rust resistance, which appears also to occur in 'Roblin'.     

Purification and partial characterization of recombinant human differentiation-stimulating factor

Recombinant human differentiation-stimulating factor (rhD-factor) has been isolated to greater than 95% purity from Chinese hamster ovary cells. RhD-factor is a glycoprotein with an apparent molecular weight of 45.6 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On gel filtration in 6 M guanidine-hydrochloride, rhD-factor elutes with an apparent molecular weight of 21.5 kDa; it elutes with an apparent molecular weight of 44.8 kDa under neutral pH (native) conditions. The amino-terminal sequence (12 residues) is consistent with the expected sequence derived from the genomic DNA sequence. Recombinant D-factor is heavily glycosylated with 30% by weight neutral sugar and 12% sialic acid. The ED50 for rhD-factor was 0.25 ng/ml. Trifluoromethanesulfonic acid-deglycosylated rhD-factor has a biological activity comparable to that of the native recombinant protein (ED50 = 0.40 ng/ml). The biological activity of rhD-factor was stable at pH 1 for 40 h, in 6 M guanidine-HCl containing buffers with or without reducing agent, and in 1% SDS. Carboxymethylation of D-factor after reduction totally destroyed biological activity.     

Purification and partial characterization of a liver cell proliferation factor called hepatopoietin

The purification and partial characterization of a liver cell proliferation factor called hepatopoietin are described. Hepatopoietin was isolated from remnant livers or blood plasma of partially hepatectomized rats and purified approximately 13,000-fold. The stokes radius was 2.65 +/- 0.2 nm and the apparent molecular weight was calculated to be 38,000 +/- 5,000 D. Hepatopoietin is a heat-stable glycoprotein and is organ specific but species nonspecific. In vivo it stimulates about three to four times the DNA synthesis as well as the mitotic rate of the liver of normal rats after i.p. injection. Hepatopoietin is inactivated upon incubation with galactosidase or trypsin-chymotrypsin.     

Purification and partial characterization of platelet-derived adherence-inhibiting factor in guinea pig

Purification and partial characterization of adherence-inhibiting factor (AIF) of platelet-granule fraction in guinea pig were studied. When freshly prepared platelet-granule fraction was subjected to a gel filtration, two neutrophil adherence-inhibiting peaks, designated AIF-I (2,800 kDa) and AIF-II (12 kDa), appeared. AIF-I was sensitive to diisopropylfluorophosphate (DFP) and originated from lysosomes, whereas AIF-II was insensitive to DEP and localized in alpha-granules. Both AIFs were released from platelets by a thrombin stimulation. As the total activity of AIF-I was about 5-fold higher than that of AIF-II, AIF-I was purified and characterized. When purified AIF-I was analyzed on SDS-polyacrylamide gel electrophoresis, the 340 kDa protein band and the other large protein band were observed. Under reducing condition, AIF-I was separated into three components (340, 190 and 165 kDa). AIF-I significantly inhibited neutrophil adherence to artificial substrata and to type IV collagen-coated plastic surface, but not to fibronectin- or plasma-coated plastic surfaces, suggesting that AIF-I inhibits neutrophil adherence not only via nonspecific adsorption sites but also via type IV collagen receptors.     

Molecular mapping of stem and leaf rust resistance in wheat

Stem rust caused by Puccinia graminis f. sp. tritici Eriks and Henn and leaf rust caused by Puccinia triticina Rob. ex Desm. are major constraints to wheat production worldwide. In the present study, F₄-derived SSD population, developed from a cross between Australian cultivars ‘Schomburgk’ and ‘Yarralinka’, was used to identify molecular markers linked to rust resistance genes Lr3a and Sr22. A total of 1,330 RAPD and 100 ISSR primers and 33 SSR primer pairs selected ob the basis of chromosomal locations of these genes were used. The ISSR marker UBC 840₅₄₀ was found to be linked with Lr3a in repulsion at a distance of 6.0 cM. Markers cfa2019 and cfa2123 flanked Sr22 at a distance of 5.9 cM (distal) and 6.0 cM (proximal), respectively. The use of these markers in combination would predict the presence or absence of Sr22 in breeding populations. A previously identified PCR-based diagnostic marker STS638 linked to Lr20 was validated in this population. This marker showed a recombination value of 7.1 cM with Lr20.     

Purification and partial characterization of serum monocytotropic factor, a platelet-derived cyclooxygenase-inducing polypeptide

It has previously been shown that a heat- and acid-stable component of human and animal sera was capable of stimulating prostanoid biosynthesis in human blood monocytes, very probably by a mechanism involving cyclooxygenase induction. Many physico-chemical characteristics of this factor are similar to those of identified platelet factors. Here we show that human platelets are a rich source of this factor (serum monocytotropic factor) and that results from experiments using arachidonic acid or thrombin as releasers are consistent with its presence in platelet membranes. Serum monocytotropic factor has been purified 1500-fold by three chromatographic steps. Purification was more difficult when starting from platelet releasates or lysates. The purified serum monocytotropic factor had an apparent molecular mass of 70,000 as judged by Sephadex G-75 chromatography and by polyacrylamide gel electrophoresis; however, when subjected to HPLC on a gel permeation column in the presence of 6 M urea, one major peak corresponding to a relative molecular mass (Mr) of 30,000-35,000 was observed, which suggests a homodimeric structure. It is therefore very likely that human platelets store, in addition to the two well-identified polypeptide growth factors, platelet-derived growth factor and transforming growth factor-beta, a third polypeptide capable of regulating prostanoid production in monocytes.     

Purification, partial characterization and biological effects of the XTC mesoderm-inducing factor

The mesoderm of Xenopus laevis is formed through an inductive interaction in which a signal from the vegetal hemisphere of the blastula acts on overlying animal pole cells. We have recently reported that the Xenopus XTC cell line secretes a mesoderm-inducing factor (MIF) which may resemble the natural signal. In this paper, we describe the purification and biological effects of XTC-MIF. XTC-MIF is a hydrophobic protein with an isoelectric point of 7.8 and an apparent relative molecular mass (Mr) of 23,500. On reduction, XTC-MIF loses its biological activity and the protein dissociates into two inactive subunits with apparent Mr of about 15,000. These properties closely resemble those of transforming growth factor type beta (TGF-beta), and it is interesting that TGF-beta 2 has recently been shown to have mesoderm-inducing activity. The biological response to XTC-MIF is graded. After exposure to 0.2-1.0 ng ml-1 XTC-MIF, stage-8 animal pole explants form mesenchyme and mesothelium. At higher concentrations, up to about 5 ng ml-1, muscle is formed, occasionally with neural tissue. In response to concentrations of XTC-MIF greater than 5-10 ng ml-1, notochord and neural tissue are usually formed. The formation of notochord and neural tissue in response to XTC-MIF represents a qualitative difference between this inducing factor and the other known group of MIFs, the heparin-binding growth factors.     

Purification and characterization of acylation stimulating protein

We have purified to homogeneity and analyzed the amino acid composition of a small (Mr 14,000), basic (pI 9.0) protein from human plasma. This has been named acylation stimulating protein (ASP) because it markedly stimulates triacylglycerol synthesis in human adipocytes. As well, it stimulates triacylglycerol synthesis in human skin fibroblasts cultured from normal individuals. Characteristic saturation curves for the cell metabolic responses to ASP were observed in both cell types with higher stimulation of oleate incorporation into triacylglycerol being observed in adipocytes. The stimulation of triacylglycerol synthesis was much greater with ASP than with insulin. Neither fatty acid binding protein nor albumin was able to mimic the ASP effect.     

Purification and partial characterization of a malignancy-associated glycoprotein

The isolation from cancer patient serum of a glycoprotein (Cc) associated with the presence of a variety of malignancies was previously reported. Although preliminary chemical and physical data indicated that Cc was different from identified circulating glycoproteins, subsequent immunological studies suggested that it was closely related to alpha 1-acid glycoprotein. Further analysis revealed the presence of two components in some Cc preparations and prompted a re-examination of the isolation and characterization data. In the present study, Cc was purified by a modified protocol which involved the use of pleural fluid obtained from individuals with cancer, and an alpha 1-acid glycoprotein antibody column to remove contaminating alpha 1-acid glycoprotein. Typically, the material not retained by the antibody column gave a single band with Mr 53,000 when subjected to sodium dodecyl sulfate-polyacrylamide electrophoresis. Amino terminal analysis revealed that the protein contained a blocked amino terminus, and carbohydrate analysis indicated that complex, asparagine-linked saccharide units were present. The product could be distinguished from alpha 1-acid glycoprotein and other previously described circulating glycoproteins by several criteria, including molecular weight, isoelectric point, and amino acid and carbohydrate composition. One of three preparations isolated had an apparent Mr of 59,000. Carbohydrate analysis as well as deglycosylation studies showed that the change in molecular weight was due to increased glycosylation.     

Surface lipids of wheat stripe rust uredospores, Puccinia striiformis, compared to those of the host

A surface lipid extract was made of uredospores of wheat stripe rust, Puccinis striiformis. The major components of the extract are β-diketones, n-alcohols and hydrocarbons. The surface lipid extract of the host wheat has a composition that is qualitatively similar, if not the same, in the major components. Even though there are quantitative differences in the two extracts, it appears that at least the three major components appear on the uredospore surface as a result of the host-parasite relationship.     

Purification and partial characterization of a stimulatory factor for lamb thymus RNA polymerase II.

A heat-stable protein (HSF) that stimulates the activity of lamb thymus RNA polymerase II has been purified 2500-fold and partially characterized. This factor stimulates the activity of RNA polymerase II up to 13 times and retains complete activity when heated at 90 degrees C for 5 min. Stimulation is observed only in the presence of RNA polymerase II and requires native DNA as template. The stimulatory factor has a sedimentation coefficient of 2.7 S, a diffusion coefficient of 9.55 x 10(-7) cm2/s, and an isoelectric point of 8.0. Calculated from the sedimentation and diffusion data, the factor has a molecular weight of about 24,000. Electrophoresis of the purified factor on polyacrylamide gels in the presence of sodium dodecyl sulfate results in a single band corresponding to a molecular weight of 25,000. The number-average length of the RNA synthesized by RNA polymerase II is increased in the presence of the factor. Sedimentation velocity and exclusion chromatography experiments suggest that the stimulatory factor interacts with RNA polymerase II. These results suggest that the factor stimulates RNA synthesis through a direct interaction with RNA polymerase II. The stoichiometry of the HSF-RNA polymerase binding appears to be about 1:1. HSF is located in the nucleus, as determined by cell fractionation studies.     

QTL characterization of resistance to leaf rust and stripe rust in the spring wheat line Francolin#1

Growing resistant wheat varieties is a key method of controlling two important wheat diseases, leaf rust and stripe rust. We analyzed quantitative trait loci (QTL) to investigate adult plant resistance (APR) to these rusts, using 141 F₅ RILs derived from the cross ‘Avocet-YrA/Francolin#1’. Phenotyping of leaf rust resistance was conducted during two seasons at Ciudad Obregon, Mexico, whereas stripe rust was evaluated for two seasons in Toluca, Mexico, and one season in Chengdu, China. The genetic map was constructed with 581 markers, including diversity arrays technology and simple sequence repeat. Significant loci for reducing leaf rust severity were designated QLr.cim-1BL, QLr.cim-3BS.1, QLr.cim-3DC, and QLr.cim-7DS. The six QTL that reduced stripe rust severity were designated QYr.cim-1BL, QYr.cim-2BS, QYr.cim-2DS, QYr.cim-3BS.2, QYr.cim-5AL, and QYr.cim-6AL. All loci were conferred by Francolin#1, with the exception of QYr.cim-2DS, QYr.cim-5AL, and QYr.cim-6AL, which were derived from Avocet-YrA. Closely linked markers indicated that the 1BL locus was the pleiotropic APR gene Lr46/Yr29. QYr.cim-2BS was a seedling resistance gene designated as YrF that conferred intermediate seedling reactions and moderate resistance at the adult plant stage in both Mexican and Chinese environments. Significant additive interactions were detected between the six QTL for stripe rust, but not between the four QTL for leaf rust. Furthermore, we detected two new APR loci for leaf rust in common wheat: QLr.cim-3BS.1 and QLr.cim-7DS.     

Purification and characterization of a factor stimulating DNA polymerase alpha activity from mouse FM3A cells

A protein factor which stimulates DNA polymerase alpha activity on heat-denatured DNA has been purified from mouse FM3A cells. The final preparation had a specific activity of 43,000 units/mg protein and lacked detectable DNA polymerase, RNA polymerase, DNA-dependent- and independent ATPase, exo- and endodeoxyribonuclease and phosphatase activities. The stimulating factor sedimented at 2.9S in a glycerol gradient. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the glycerol gradient fraction revealed the presence of a major band of 36,000 daltons, the amount of which corresponded well with the level of stimulating activity. The stimulation by the factor was specific for heat-denatured DNA, and a little or no stimulation was observed with native DNA, ribo- and deoxyribohomopolymers and single stranded circular DNA. Alkaline sucrose gradient sedimentation analysis of the reaction products revealed that newly synthesized DNA was covalently linked to the termini of heat-denatured DNA. The average chain length of the elongated span determined by the digestion with micrococcal nuclease and phosphodiesterase II, did not differ between in the presence and absence of the stimulating factor, suggesting that the stimulation by the factor was due to the increase in the initiation frequency of DNA synthesis from the 3'-hydroxyl terminus of heat-denatured DNA.     

Purification and characterization of a ribosome dissociation factor (eukaryotic initiation factor 6) from wheat germ.

A wheat germ ribosome dissociation factor, eukaryotic initiation factor 6 (eIF-6), has been purified almost to homogeneity from the 25 to 40% ammonium sulfate fraction of the postribosomal supernatant. This dissociation factor is distinct from initiation factor eIF-3 and its chromatographic properties permit its separation from the known wheat germ initiation factors. Under certain conditions, eIF-6 stimulates the incorporation of amino acids into polypeptides in a partially fractionated wheat germ cell-free system. The eight-step purification procedure developed includes chromatography on DEAE-cellulose, phosphocellulose, Sephadex G-75, and hydroxyapatite and yields a dissociation factor more than 80% pure. The purified factor is composed of a single polypeptide chain with a molecular weight of approximately 23,000 as determined by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is an acidic protein which is heat labile and is inactivated by treatment with N-ethylmaleimide. The dissociation factor is much more effective in preventing the reassociation of 40 S and 60 S ribosomal subunits than in directly dissociating 80 S ribosomes. Like Escherichia coli IF-3, about 10 pmol of the dissociation factor are required to dissociate 1 pmol of ribosomes.     

Purification and partial characterization of a dipeptidase from barley

A peptidase hydrolyzing the dipeptide Ala-Gly optimally at pH 8 to 9 was purified about 3500-fold from germinated grains of barley (Hordeum vulgare L.). According to disc electrophoresis in the presence of sodium dodecyl sulfate, the preparation was about 90% pure.