Physical map of DNA from a new cauliflower mosaic virus strain.

The restriction enzymes AluI, BamHI, BglII, EcoRI, HindIII, and SalI have been used to characterize and map a new cauliflower mosaic virus strain (Cabb-S). These fragments have been ordered by examining their overlapping regions after double enzymatic digestion. The single SalI cleavage site was chosen as the point of origin. We compare this strain with those already described.

Restriction map of native and cloned cauliflower mosaic virus DNA.

Cloned CaMV DNA replicates faithfully in Escherichia coli, since the restriction map of the cloned DNA can be superimposed over that of the native viral DNA. However, some short fragments were difficult to detect in the restricted native viral DNA, whereas they formed clear bands when derived from cauliflower mosaic virus (CaMV) DNA clones propagated in the E. coli host. Apparently, the small fragments that carry variable-length single-stranded gaps present only in native viral DNA, give rise to diffuse weak bands difficult to recognize in gels. Comparison of maps for several CaMV strains permits evaluation of their possible evolutionary relationship.

Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants.

Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals.

Production and Characterization of Monoclonal Antibodies against NADPH-Cytochrome P-450 Reductases from Helianthus tuberosus.

Monoclonal antibodies (mAbs) against a plant NADPH-cytochrome P-450 (Cyt P-450) reductase from Jerusalem artichoke (Helianthus tuberosus) tuber were prepared. These antibodies were produced by hybridoma resulting from the fusion of spleen cells from a rat immunized with a purified preparation of the reductase and mouse myeloma cells. The mAbs thus obtained were screened for their interaction with the reductases, first in western dots and then in blots, and for their ability to inhibit the NADPH-cytochrome c (Cyt c) reductase activity from Jerusalem artichoke microsomes. Among the 11 clones giving a positive response on western blots, only 6 were also able to inhibit microsomal NADPH-Cyt c reductase activity, and the microsomal Cyt P-450 monooxygenase activities dependent upon electrons transferred by the reductase. Thus, two families of mAbs were characterized: a family of mAbs that interact with epitopes of the reductase implicated in the reduction of Cyt P-450 by NADPH (binding sites for NADPH, flavin mononucleotide, flavin adenine dinucleotide, and Cyt P-450), and a structural family, whose members recognize epitopes outside the active site of the reductases. These mAbs specifically recognize the reductase, and all of them interact with all of the isoforms, indicating that important primary or secondary structural analogies exist between the isoforms, not only at the active site, but also at the level of epitopes not directly associated with catalytic activity.

Multiple forms of NADPH-cytochrome P450 reductase in higher plants.

We report on the presence of multiple forms of NADPH-cyt P450 reductase in microsomes from higher plants. This contrasts with the animal cyt P450 monooxygenases, where the numerous cyt P450 isoforms are reduced by a single form of reductase. Three NADPH-cyt c reductases have been resolved from Jerusalem artichoke tuber microsomes by chromatography on Reactive Red Agarose and Concanavalin A-Sepharose. Their molecular weights, determined by sodium dodecylsulfate-gel electrophoresis, are 80,000, 82,000 and 84,000. The three proteins share common epitopes and are dependent upon FMN for catalytic activity. They are highly selective for NADPH as electron donor, and allowed effective reconstitution of trans-cinnamic acid and 3,9-dihydroxypterocarpan 6a-hydroxylase activities with purified cyt P450 fractions from Helianthus tuberosus and Glycine max, respectively. As such, they appear as true isoenzyme forms of NADPH-cyt P-450 reductase.

omega-Hydroxylation of Z9-octadecenoic, Z9,10-epoxystearic and 9,10-dihydroxystearic acids by microsomal cytochrome P450 systems from Vicia sativa.

A microsomal fraction from etiolated Vicia sativa seedlings incubated aerobically with [1-14C]oleic acid (Z9-octadecenoic acid) or [1-14C]9,10-epoxystearic acid or [1-14C]9,10-dihydroxystearic acid catalyzed the NADPH-dependent formation of hydroxylated metabolites. The chemical structure of compounds formed from oleic, 9,10-epoxystearic or 9,10-dihydroxystearic acids was established by gas chromatography/mass spectra analysis to be 18-hydroxyoleic acid, 18-hydroxy-9,10-epoxystearic acid and 9,10,18-trihydroxystearic acid, respectively. The reactions required O2 and NADPH and were inhibited by carbon monoxide. As expected for monooxygenase reactions involving cytochrome P450, inhibition could be partially reversed by light and all three reactions were inhibited by antibodies raised against NADPH-cytochrome P450 reductase from Jerusalem artichoke. The omega-hydroxylation of the three substrates was enhanced in microsomes from clofibrate induced seedlings.

Monospecific polyclonal antibodies directed against purified cinnamate 4-hydroxylase from Helianthus tuberosus. Immunopurification, immunoquantitation, and interspecies cross-reactivity.

We recently reported the purification of cinnamic acid 4-hydroxylase (CA4H), a cytochrome P-450 catalyzing the second reaction of the general phenylpropanoid pathway, from Jerusalem artichoke (Helianthus tuberosus L.) (B. Gabriac, D. Werck-Reichhart, H. Teutsch, F. Durst [1991] Arch Biochem Biophys 288: 302-309). Rabbit polyclonal antibodies were raised against the native and denaturated nitrocellulose-bound enzyme. Only the immunoglobulins G (IgGs) elicited upon immunization with native enzyme produced strong inhibition of catalytic activity and good cross-reactivity on western blots. In microsomes from H. tuberosus tissues induced by wounding and various chemicals, a positive correlation between catalytic activity and amounts of immunoreactive protein on western blots was observed. When coupled to cyanogen bromide-activated Sepharose, purified IgGs selectively retained CA4H activity from solubilized plant microsomes. Acid elution from the immunoaffinity matrix provided a rapid procedure for high-yield purification of the CA4H protein. The same IgGs immunoprecipitated a single protein from the in vitro translation products of mRNA isolated from wounded tissues. The apparent molecular weight (57,000) of this polypeptide was identical to that of CA4H purified from tuber microsomes. Immunochemical relatedness between CA4H from different plant species was demonstrated by strong inhibition of catalytic activity and immunopurification of several orthologous enzymes, using IgGs directed against CA4H from H. tuberosus. However, only limited interspecies cross-reactivity was observed on western blots. A careful immunochemical analysis indicates that CA4H immunoreactivity significantly differs from plant to plant. Results are discussed in terms of antibody specificity, enzyme glycosylation, and CA4H regulation.

Functional expression in yeast and characterization of a clofibrate-inducible plant cytochrome P-450 (CYP94A1) involved in cutin monomers synthesis.

The chemical tagging of a cytochrome P-450-dependent lauric acid omega-hydroxylase from clofibrate-treated Vicia sativa seedlings with [1-14C]11-dodecynoic acid allowed the isolation of a full-length cDNA designated CYP94A1. We describe here the functional expression of this novel P-450 in two Saccharomyces cerevisiae strains overproducing their own NADPH-cytochrome P-450 reductase or a reductase from Arabidopsis thaliana. The results show a much higher efficiency of the yeast strain overproducing the plant reductase compared with the yeast strain overproducing its own reductase for expressing CYP94A1. The methyl end of saturated (from C-10 to C-16) and unsaturated (C18:1, C18:2 and C18:3) fatty acids was mainly oxidized by CYP94A1. Both E/Z and Z/E configurations of 9, 12-octadecadienoic acids were omega-hydroxylated. Lauric, myristic and linolenic acids were oxidized with the highest turnover rate (24 min-1). The strong regioselectivity of CYP94A1 was clearly shifted with sulphur-containing substrates, since both 9- and 11-thia laurate analogues were sulphoxidized. Similar to animal omega-hydroxylases, this plant enzyme was strongly induced by clofibrate treatment. Rapid CYP94A1 transcript accumulation was detected less than 20 min after exposure of seedlings to the hypolipidaemic drug. The involvement of CYP94A1 in the synthesis of cutin monomers and fatty acid detoxification is discussed.

Exploitation of parasite derived antigen in therapeutic success of human cutaneous leishmaniasis in Brazil.

In a complete study in 25 patients with American cutaneous leishmaniasis, caused by Leishmania braziliensis complex, immunotherapeutic efficacy of parasite derived antigen (94-67 KD) has been compared to antimonial therapy. Additionally, to delineate the mechanism of therapeutic success, microscopical features of immune response in active lesions and healed or non-healed lesions following therapy were analyzed. The results showed that cure rates in immunotherapy and chemotherapy were equal (> 83%). The immunohistochemical changes in two therapeutic groups were also largely similar. The analysis of humoral and cellular immune response suggest that appropriate stimulation of T helper cells in the lesion site, in association with one or more cytokines, play a key role in the healing process.

Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway.

Cinnamate 4-hydroxylase [CA4H; trans-cinnamate,NADPH:oxygen oxidoreductase (4-hydroxylating), EC 1.14.13.11] is a cytochrome P450 that catalyzes the first oxygenation step of the general phenylpropanoid metabolism in higher plants. The compounds formed are essential for lignification and defense against predators and pathogens. We recently reported the purification of this enzyme from Mn(2+)-induced Jerusalem artichoke (Helianthus tuberosus L.) tuber tissues. Highly selective polyclonal antibodies raised against the purified protein were used to screen a lambda gt11 cDNA expression library from wound-induced Jerusalem artichoke, allowing isolation of a 1130-base-pair insert. Typical P450 domains were identified in this incomplete sequence, which was used as a probe for the isolation of a 1.7-kilobase clone in a lambda gt10 library. A full-length open reading frame of 1515 base pairs, encoding a P450 protein of 505 residues (M(r) = 57,927), was sequenced. The N terminus, essentially composed of hydrophobic residues, matches perfectly the microsequenced N terminus of the purified protein. The calculated pI is 9.78, in agreement with the chromatographic behavior and two-dimensional electrophoretic analysis of CA4H. Synthesis of the corresponding mRNA is induced in wounded plant tissues, in correlation with CA4H enzymatic activity. This P450 protein exhibits the most similarity (28% amino acid identity) with avocado CYP71, but also good similarity with CYP17 and CYP21, or with CYP1 and CYP2 families. According to current criteria, it qualifies as a member of a new P450 family.