Purification of the cyclooxygenase that forms prostaglandins. Demonstration of two forms of iron in the holoenzyme

The fatty acid cyclooxygenase (ec 1.14.99.1) that produces the prostaglandin and thromboxane precursor, 15-hydroperoxy-9 alpha, 11 alpha-peroxidoprosta-5, 13-dienoic acid (PGG2), has been purified from sheep vesicular glands to a specific activity of 46,000 units/mg of protein by combining detergent solubilization, (NH4)SO4 fractionation, chromatography on DEAE-cellulose and Flurbiprofen-Sepharose, isoelectric focusing, and gel filtration. The final enzyme preparation exhibited only one band of 70,000 molecular weight following sodium dodecyl sulfate gel electrophoresis and staining with Coomassie blue. Treatment of the purified oxygenase with [3H] acetylsalicylic acid yielded a radioactive product which co-electrophoresed with the protein of 70,000 molecular weight. Thus, the isolated protein appeared to be the same one which, in crude preparations, selectively binds acetyl groups in association with prostaglandin synthetic activity. Incubation of the purified oxygenase with [1-14C] arachidonic acid in the presence of stannous chloride yielded only 9 alpha, 11 alpha, 15-trihydroxy-prosta-5,13-dienoic acid (PGF2alpha). Without stannous chloride, a mixture of radioactive products was observed which was characteristic of nonenzymic breakdown of PGG2. Thus, the isolated enzyme catalyzed the insertion of both oxygen molecules required for the formation of prostaglandins and thromboxanes from polyunsaturated fatty acid substrates. The aerobic absorption spectrum of the isolated oxygenase showed a faint peak at 412 nm indicative of heme. The iron content indicated that a significant amount of nonheme iron was present. The purified oxygenase was activated by added hemin, which was readily bound to the protein. The subsequently isolated heme-protein complex showed a major absorption peak at 407 nm.     

Purification and characterization of a highly stable cysteine protease from the latex of Ervatamia coronaria

A highly stable cysteine protease was purified to homogeneity from the latex of Ervatamia coronaria by a simple purification procedure involving ammonium sulfate precipitation and ion-exchange chromatography. The molecular mass was estimated to be approximately 25,000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon 280 nm 1%) of the enzyme was 24.6. The enzyme hydrolyzed denatured natural substrates like casein, hemoglobin, azoalbumin, and azocasein with a high specific activity but showed low specific activity towards synthetic substrates. The pH and temperature optima were 7.5-8.0 and 50 degrees C respectively. The activity of the enzyme was strongly inhibited by thiol-specific inhibitors like leupeptin, iodoacetamide, PCMB, NEM, and mercuric chloride. The striking property of this enzyme was its stability over a wide pH range (2-12) and other extreme conditions of temperature, denaturants, and organic solvents. The N-terminal sequence showed marked similarity to known cysteine proteases.     

Characterization of an activating factor required for hydrolysis of Gm2 ganglioside catalyzed by hexosaminidase A

Separation of the hexosaminidase A (EC 3.2.1.52) and B isozymes of human liver by ion-exchange chromatography results in recovery of greater than 80% of the activity in crude extracts when synthetic substrates are used to monitor enzyme activity. Only 15% of hexosaminidase activity toward the N-acetylgalactosaminyl (N-acetylneuraminyl) galactosyl glucosylceramide (Gm2 ganglioside) substrate is recovered and all of this activity is associated with the hexosaminidase A Fraction. The low level of Gm2 ganglioside hydrolase activity in the hexosaminidase A fraction could be enhanced by coincubation with column fractions which contain hexosaminidase B. The activating factor, which has been partially purified by gel filtration, is a heat-stable protein with a molecular weight of 36 000 and is without enzyme activity toward hexosaminidase substrates. Highly purified hexosaminidase A or crude hexosaminidase A recovered after gel filtration on Sephadex G-100 has no Gm2 ganglioside hydrolase activity. The Gm2 ganglioside hydrolase activity of these hexosaminidase. A preparations can be completely restored by addition of activating factor. The activating factor does not affect the rate of hydrolysis of synthetic substrate or asialo Gm2 ganglioside catalyzed by hexosaminidase A.     

Epstein-Barr virus (EBV) and Hodgkin''s disease in a multi-ethnic population in Malaysia

The plant pathogenic fungus Verticillium dahliae produced extracellular alkaline protease activity when grown in liquid medium supplemented with a protein source. A serine protease was purified 80-fold in a single step, using cation-exchange chromatography, from the filtrate of cultures grown with skim milk as a protein source. N-terminal amino acid sequence analysis of the 30-kDa protein (VDP30) that copurified with the serine protease activity suggested that VDP30 is a trypsin-like protein. The purified enzyme hydrolyzed the synthetic substrate N alpha-benzoyl-DL-arginine p-nitroanilide hydrochloride (BAPNA), and the activity on BAPNA was inhibited by leupeptin, further verifying the trypsin-like nature of the enzyme.     

Purification of a protein phosphatase from chloroplast stroma capable of dephosphorylating the light-harvesting complex-II

A protein phosphatase was purified from the stroma of Pea (Pisum sativum L.) chloroplasts that is capable of dephosphorylating synthetic phosphopeptides. Following chromatographic purification of greater than 400-fold, two-dimensional electrophoresis indicated that the stromal protein phosphatase is a 29-kD protein. A similar molecular size was determined for the protein-phosphatase activity using gel-permeation chromatography, indicating that the stromal protein phosphatase is probably a monomer. The purified enzyme was able to dephosphorylate synthetic phosphopeptides, which mimic the thylakoid light-harvesting complex II (LHC-II) N terminus, as well as LHC-II in thylakoid membranes, but did not dephosphorylate the major 64-kD phosphoprotein in the stroma. The stromal protein phosphatase did not discriminate between dephosphorylation of phosphothreonine and phosphoserine residues in synthetic peptide substrates, providing further evidence that this enzyme is distinct from the protein phosphatase localized in thylakoid membranes. The exact physiological role of the stromal protein phosphatase has yet to be determined, but it may function in the dephosphorylation of LHC-II.     

Purification and characterization of a prolidase from Aureobacterium esteraromaticum

An EDTA-insensitive prolidase (proline dipeptidase, EC 3.4.13.9) was isolated from a cell-free extract of Aureobacterium esteraromaticum IFO 3752. The enzyme was purified almost to homogeneity using acetone precipitation, hydrophobic chromatography, ion-exchange chromatography, and gel-permeation chromatography. The enzyme has a molecular weight of about 440,000 by gel permeation chromatography, and about 40,000 by SDS polyacrylamide gel electrophoresis. The isoelectric point was 4.6. The enzyme hydrolyzed aminoacylprolines such as Ser-Pro. Thr-Pro, Gly-Pro, Ala-Pro, Ile-Pro, Leu-Pro, and Pro-Pro. It also hydrolyzed Gly-Hyp and Pro-Hyp. The rate of hydrolysis for Pro-Hyp was the highest among the substrates tested. Optimum pH for hydrolyzing Pro-Hyp was 9.0 and the enzyme was stable in the pH range from 5 to 10. The optimum temperature was estimated to be 45 degrees C using 10 min of reaction. At least 90% of the initial activity remained after 30 min of incubation at 60 degrees C. p-Chloromercuribenzoic acid and o-phenanthrolin inhibited the enzyme's activity while EDTA did not. Addition of Mn2+ ion did not stimulate activity. These results suggest either that the metal ion in the enzyme may be tightly bound to the polypeptide chain, or that the enzyme is not a metallo-enzyme but a thiol-enzyme.     

Biochemical characterization of basilase, a fibrinolytic enzyme from Crotalus basiliscus basiliscus

Snake venoms, especially from the Crotalidae family, contain a variety of enzymes that prevent blood coagulation by virtue of their fibrinolytic enzymes. Nineteen snake venoms were screened for fibrinolytic activity and the highest activity was found in the venom of Crotalus basiliscus basiliscus venom. The active principle, basilase, was isolated, purified, and found to have fibrinolytic and fibrinogenolytic activity. It had a molecular weight of 22,000 and 1 mol of zinc per mole of protein associated with it. The proteolytic activity of the enzyme against dimethyl casein was inhibited by ethylenediaminetetraacetic acid and alpha 2-macroglobulin. It did not inactivate alpha 2-macroglobulin. Basilase did not have any of the following activities: thrombin-like, factor X-like, protein C activating, or urokinase-like. It caused neither hemorrhage nor platelet aggregation. In spite of its proteolytic activity, basilase did not hydrolyze the membranes of platelets. Basilase had 24% alpha-helix, 31% beta-sheet, 25% turns, and 20% unordered structure, as determined by Fourier Transform Infrared spectroscopy. Basilase is an enzyme that hydrolyzes fibrin directly without activation of plasminogen.     

Purification and characterization of laccase from Chaetomium thermophilium and its role in humification

Chaetomium thermophilium was isolated from composting municipal solid waste during the thermophilic stage of the process. C. thermophilium, a cellulolytic fungus, exhibited laccase activity when it was grown at 45 degreesC both in solid media and in liquid media. Laccase activity reached a peak after 24 h in liquid shake culture. Laccase was purified by ultrafiltration, anion-exchange chromatography, and affinity chromatography. The purified enzyme was identified as a glycoprotein with a molecular mass of 77 kDa and an isoelectric point of 5.1. The laccase was stable for 1 h at 70 degreesC and had half-lives of 24 and 12 h at 40 and 50 degreesC, respectively. The enzyme was stable at pH 5 to 10, and the optimum pH for enzyme activity was 6. The purified laccase efficiently catalyzed a wide range of phenolic substrates but not tyrosine. The highest levels of affinity were the levels of affinity to syringaldazine and hydroxyquinone. The UV-visible light spectrum of the purified laccase had a peak at 604 nm (i.e., Cu type I), and the activity was strongly inhibited by Cu-chelating agents. When the hydrophobic acid fraction (the humic fraction of the water-soluble organic matter obtained from municipal solid waste compost) was added to a reaction assay mixture containing laccase and guaiacol, polymerization took place and a soluble polymer was formed. C. thermophilium laccase, which is produced during the thermophilic stage of composting, can remain active for a long period of time at high temperatures and alkaline pH values, and we suggest that this enzyme is involved in the humification process during composting.     

Purification and characterization of a membrane-bound ATPase from Acetabularia cliftonii that corresponds to a Cl(-)-translocating ATPase in Acetabularia acetabulum

A Mg(2+)-ATPase was solubilized from membranes of Acetabularia cliftonii using nonanoyl-N-methylgluconamide and purified by ion-exchange and gel permeation chromatography. One active ATPase fraction after Mono Q chromatography had a specific activity of 10 units/mg of protein. Judged from subunit composition [54 (a), 50 (b) with a fainter band around 40 kDa], catalytic properties, and N-terminal amino acid sequence of the b subunit, the isolated enzyme was comparable to the Cl(-)-ATPase of Acetabularia acetabulum. Immunological characterization of both subunits showed significant similarity to the F type of ATPase. Cl(-)-transport activity was observed by reconstitution studies into liposomes.     

Role of metalloproteinases in the development and healing of acetic acid-induced gastric ulcer in rats

The three main components involved in thrombosis and haemostasis are thrombin, platelets, and plasmin. Almost all inhibitors of thrombosis are focused either on the inhibition of thrombin or on the inhibition of platelets. We designed a construct using the fibrinolytic activity of staphylokinase, fused via a cleavable linker to an antithrombotic peptide of 29 amino acids. The peptide was designed to include three inhibitory regions: (1) the Arg-Gly-Asp (RGD) amino acid sequence to prevent fibrinogen binding to platelets; (2) a part of fibrinopeptide A, an inhibitor of thrombin; and (3) the tail of hirudin, a potent direct antithrombin. The amino acid sequence of the 29 amino acid peptide was reverse translated, and the gene was chemically synthesised and cloned into an expression vector as a 3' fusion to the staphylokinase gene. Gene expression was induced in E. coli Top 10 cells and the fusion protein, designated PLATSAK, was purified using metal affinity chromatography. The purified fusion protein significantly lengthened the activated partial thromboplastin time and thrombin time and inhibited the amidolytic activity of thrombin. The fibrinolytic activity was almost equal to that of recombinant staphylokinase as measured with a thrombelastograph. Platelet aggregation was not markedly inhibited by PLATSAK, probably due to the unfavourable three dimensional structure, with the Arg-Gly-Asp sequence buried inside. Our results confirm that it is feasible to design and produce a hybrid multifunctional protein that targets various components of the haemostatic process.     

Rabbit red blood cell hexokinase. Purification and properties

Rabbit red blood cell hexokinase (EC 2.7.1.1.) has been purified 300,000-fold by a combination of ion exchange chromatography, affinity chromatography, and preparative polyacrylamide gel electrophoresis. The hexokinase activity has been isolated in 35% yield as a protein that is homogeneous by polyacrylamide and sodium dodecyl sulfate gel electrophoresis. The highest specific activity obtained was 145 units/mg of proteins. The native protein has a molecular weight of 110,000 by gel filtration on Ultrogel AcA 44 and 112,000 by sedimentation velocity on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gels gave a molecular weight of 110,000 indicating that hexokinase is a monomer. The enzyme had a pI of 6.20 to 6.30 pH units by isoelectric focusing. The enzyme was specific for Mg . ATP and Mg . ITP as the nucleotide substrates. Several hexokinase with different affinities.     

A new peptide-N4-(acetyl-beta-glucosaminyl)asparagine amidase from soybean (Glycine max) seeds: purification and substrate specificity

We report here the isolation and characterization of a peptide-N4-(acetyl-beta-glucosaminyl) asparagine amidase (peptide: N-glycanase) from soybean (Glycine max) seeds. The enzyme was purified to homogeneity with 6.5% yield from defatted soybean meal extract by ion-exchange chromatography, gel filtration, hydroxyapatite chromatography, and hydrophobic chromatography. The purified enzyme, designated PNGase-GM, had the apparent molecular mass of 93 kDa by SDS-PAGE and 90 kDa by gel filtration, indicating this PNGase is a monomeric protein. The enzyme showed maximal activity at pH 4.5-5.0. PNGase-GM was capable of hydrolyzing the beta-aspartylglycosylamine linkage (GlcNAc beta 1-->Asn) of various glycopeptide substrates bearing high-mannose type, hybrid type, and xylose/fucose-containing plant complex type N-glycan units, while this amidase was far less active on the glycopeptides bearing sialylated animal complex-type glycans.     

Cleavage activity of hepatitis C virus serine proteinase

To study the character of the hepatitis C virus (HCV) encoding serine proteinase and to search for inhibitors, a practical in vitro assay system using the purified enzyme and synthetic peptide substrates was established. The enzyme used was expressed in Escherichia coli as a fusion form with protein tags and purified to apparent homogeneity by single-step affinity chromatography. The purified enzyme exhibited proteolytic activity with pH optima of around eight, and the addition of NS4A fragments increased the activity as well as the thermal stability of the enzyme. The activity was inhibited by EDTA and some divalent ions, i.e., copper and zinc, though calcium, magnesium, and manganese were stimulative both in the presence and absence of the NS4A fragment. None of the common protease inhibitors, including serine protease inhibitors, effectively inhibited the activity. Based on the kinetic parameters of the cleavage reaction of the synthetic 20 mer peptides corresponding to the three cleavage sites, NS4A/4B, NS4B/5A, and NS5A/5B, the peptide with the NS5A/5B junction was found to be the most efficient substrate. Analysis of the minimal peptide substrate of NS5A/5B indicated that 5 to 7 amino acids on both sides of the junction were required for efficient cleavage. These findings are expected to contribute to the search for a proteinase inhibitor.     

Construction and expression of a synthetic streptavidin-encoding gene in Escherichia coli

A synthetic gene encoding 'core' streptavidin (SAV) [amino acid (aa) residues 13-140 of Streptomyces avidinii SAV] has been efficiently expressed in Escherichia coli from the IPTG-inducible lac promoter of plasmid pET3a. In this system, expression levels are nearly tenfold greater for the synthetic gene than for the corresponding native gene. The synthetic gene was constructed from overlapping oligodeoxyribonucleotides whose sequences were optimized to incorporate codons preferred by highly expressed E. coli genes. Biochemical characterizations by gel methods, aa analysis, N-terminal sequencing, and size exclusion chromatography show that the synthetic gene product purified by affinity chromatography possesses the properties expected for core SAV.     

Differences of microvascular endothelium in the bovine corpus luteum of pregnancy and the corpus luteum of the estrous cycle

A haemolysin produced by Actinomyces pyogenes ATCC 8164 was purified from culture supernatant by ammonium sulphate and polyethylene glycol precipitation, ion-exchange chromatography on DEAE-Sephacel, and fast-protein-liquid-chromatography on Superose 12 prep grade. The purified haemolysin, designated as pyolysin, displayed a single band on poly-acrylamide gel electrophoresis, indicating a molecular weight of 55000. Additionally, using gel filtration, the same molecular weight was estimated. Further studies of the eluate of ion-exchange chromatography using isoelectric focusing also revealed a single protein band at pH 9.38 with haemolytic activity. A specific antiserum produced against pyolysin inhibited the haemolytic activity. The purity of the isolated protein was also determined by Western Blot analysis with antiserum obtained from a cow inoculated with culture supernatant from A. pyogenes and Peptococcus indolicus. The isolated pyolysin appeared to be heat-labile and displayed cytotoxic effects on poly-morphonuclear leucocytes and on pTK2 kidney cells.     

Purification of glutamine transaminase K/cysteine conjugate beta-lyase from rat renal cytosol based on hydrophobic interaction HPLC and gel permeation FPLC

Cysteine conjugate beta-lyase (beta-lyase, EC 4.4.1.13) was purified to homogeneity from rat renal cytosol using a new and highly efficient method, based on C3-hydrophobic interaction (HI) high-performance liquid chromatography (HPLC) in combination with gel permeation fast protein liquid chromatography. The purity of the enzyme was judged from SDS-PAGE and C18-reversed-phase HPLC. The beta-lyase was estimated to be a homodimer consisting of a 47,400-Da subunit with absorption maxima at 280 and 420-430 nm. The specific activity of the purified beta-lyase toward S-(1,2-dichlorovinyl)-L-cysteine (1,2-DCVC) in the presence of alpha-keto-gamma-methiolbutyric acid (KMB) was 6.4 mumol/min/mg protein, which is by far the highest value so far reported. Kinetic analysis of 1,2-DCVC metabolism by the enzyme in the presence of KMB gave Km and Vmax values of 0.33 mM and 8.4 mumol/min/mg protein, respectively. No significant activity of the purified enzyme was detectable with S-2-benzothiazolyl-L-cysteine up to 2 mM. The purified enzyme also had glutamine transaminase K activity (EC 2.6.1.64) as assayed with phenylalanine and KMB as substrates. This specific activity was 16.0 mumol/min/mg. Amino acid analysis of the purified beta-lyase was carried out and was found to be closely similar to the amino acid composition of five other pyridoxal phosphate (PLP)-containing amino acid amino-transferases. This suggests that glutamine transaminase K/cysteine conjugate beta-lyase is a typical member of the PLP-containing aminotransferase group.     

Growth-regulated formation of heteromeric complexes of the centromere and promoter factor, Cbf1p, in yeast

The gastrointestinal pathogen Aeromonas hydrophila strain A186 produces a collagen-binding protein (CNBP) which is found extracellularly and loosely associated with the cell surface. The cell-associated CNBP was purified by sequential ammonium sulphate precipitation, size-exclusion chromatography and ion-exchange chromatography, or by sequential ammonium sulphate precipitation and affinity chromatography with collagen-Sepharose. The purified CNBP was homogeneous in SDS-PAGE, and had a mol. wt of c. 98 kDa. Cyanogen bromide cleavage of the CNBP destroyed collagen-binding activity; however, enzymic digestion with Staphylococcus aureus V8 protease generated > 10 polypeptide fragments, from which a 30-kDa polypeptide contained the strongest collagen-binding activity. Binding of collagen by the CNBP was restricted to the alpha1 (I) chain of the collagen molecule and binding seemed to involve both the carbohydrate moieties and certain peptide sequences on the collagen. Collagen-saccharides generated by alkaline hydrolysis inhibited collagen binding by A. hydrophila. Also, glycosidase digestion and chemical alteration of the carbohydrate residues of collagen reduced its ability to be bound by the CNBP. Collagen-homologous synthetic peptides inhibited binding of 125I-collagen by the bacteria.     

cdc2-like kinase from rat spinal cord specifically phosphorylates KSPXK motifs in neurofilament proteins: isolation and characterization

A protein kinase that phosphorylates a specific KSP sequence [K(S/T)PXK], which is abundant in high molecular weight neurofilament (NF) proteins, was identified and isolated from rat spinal cord. Characterization of this enzyme activity revealed a close relationship with p34cdc2 kinase with respect to its molecular mass (32.5 kDa by SDS/PAGE) and substrate specificities. It could phosphorylate a synthetic peptide analog of the simian virus 40 large tumor antigen, reportedly a specific substrate for p34cdc2 kinase. Histone (H1) and peptide analogs of the KSP sequence present in the C-terminal end of rat and mouse neurofilament proteins were phosphorylated. This kinase did not phosphorylate alpha-casein and peptide substrates of other known second messenger-dependent or -independent kinases. Dephosphorylated rat NF protein NF-H was strongly phosphorylated by the purified enzyme; NF proteins NF-M and native NF-H, but not NF-L, were slightly phosphorylated. Studies on synthetic peptide analogs of KSP repeats with substitution of specific residues, known to be present in the C-terminal regions of NF-H, revealed a consensus sequence of X(S/T)PXK, characteristic of the p34cdc2 kinase substrate. On Western blots, the enzyme was immunoreactive with antibody against the C-terminal end of cdc2 kinase (mouse) and neuronal cdc2-like kinase from rat but not with an antibody against the conserved PSTAIRE region of the p34cdc2 kinase. The antibody against the C-terminal end of cdc2 kinase could immunoprecipitate (immunodeplete) the purified kinase activity. Since the adult nervous system is composed primarily of postmitotic cells, the present observations indicate a nonmitotic role for this cdc2-like kinase activity. The effective phosphorylation of NF-H by this kinase suggests a function in axonal structure.     

Structure/function analyses of recombinant variants of human factor Xa: factor Xa incorporation into prothrombinase on the thrombin-activated platelet surface is not mimicked by synthetic phospholipid vesicles

This report describes the expression, purification, and characterization of a series of recombinant factor Xa variants bearing aspartate substitutions for each of the glutamate residues which normally undergo gamma-carboxylation. Factor X was expressed in human embryonic kidney cells and purified from conditioned media by immunoaffinity and hydroxylapatite chromatography. Factor X was activated with Russell's viper venom factor X activator, and single-chain unactivated factor X was removed from activated factor X by size-exclusion chromatography. Recombinant wild-type factor Xa had normal activity in a clotting assay, and mutants with aspartate substitutions for glas residues 16, 26, and 29 had no detectable clotting activity. In purified component assays, these gla variants had essentially no detectable activity in the prothrombinase complex assembled on synthetic phospholipid vesicles but had significant activity when the prothrombinase was assembled on thrombin-activated platelets. In addition, the gla 32 variant had normal activity in the platelet prothrombinase but diminished activity in prothrombinase assembled on synthetic PSPC vesicles. These differences were not accounted for by the total phospholipid composition of the thrombin-activated platelet membrane. We have produced fully active recombinant human factor Xa and demonstrated that gla residues 16, 26, and 29 are critical for normal activity of factor Xa. More importantly, this study provides an extensive characterization of macromolecular enzyme complex formation with gla variants of a vitamin K-dependent coagulation protein and provides evidence that prothrombinase complex assembly on thrombin-activated platelets is not equivalent to assembly on synthetic phospholipid vesicles. The data suggest that thrombin-activated platelets possess some element(s) (other than 30% phosphatidyl serine or factor Va), presumably either protein or phospholipid, that serves as a component of the factor Xa binding site.     

Purification, characterization, and antifungal activity of chitinase from Fusarium chlamydosporum, a mycoparasite to groundnut rust, Puccinia arachidis

Chitinase (EC 3.2.1.14) was isolated from the culture filtrate of Fusarium chlamydosporum and purified by ion-exchange chromatography and gel filtration. The molecular mass of purified chitinase was 40 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chitinase was optimally active at a pH of 5 and stable from pH 4 to 6 and up to 40 degrees C. Among the metals and inhibitors tested, mercuric chloride completely inhibited the enzyme activity. The activity of chitinase was high on colloidal and pure chitin. The purified chitinase inhibited the germination of uredospores of Puccinia arachidis and also lysed the walls of uredospores and germ tubes. The results from these experiments indicated that chitinase of F. chlamydosporum plays an important role in the biocontrol of groundnut rust.