Ouchterlony Double Diffusion Test Research Articles

Inhibition of local effects induced by Bothrops erythromelas snake venom: Assessment of the effectiveness of Brazilian polyvalent bothropic antivenom and aqueous leaf extract of Jatropha gossypiifolia

Bothrops erythromelas is a snake of medical importance responsible for most of the venomous incidents in Northeastern Brazil. However, this species is not included in the pool of venoms that are used in the Brazilian polyvalent bothropic antivenom (BAv) production. Furthermore, it is well known that antivenom therapy has limited efficacy against venom-induced local effects, making the search for complementary alternatives to treat snakebites an important task. Jatropha gossypiifolia is a medicinal plant widely indicated in folk medicine as an antidote for snakebites, whose effectiveness against Bothrops jararaca venom (BjV) has been previously demonstrated in mice. In this context, this study assessed the effectiveness of the aqueous extract (AE) of this plant and of the BAv against local effects induced by B. erythromelas venom (BeV). Inhibition of BeV-induced edematogenic and hemorrhagic local effects was assayed in mice in pre-treatment (treatment prior to BeV injection) and post-treatment (treatment post-envenomation) protocols. Inhibition of proteolytic, phospholipase A2 (PLA2) and hyaluronidase enzymatic activities of BeV were evaluated in vitro. BAv cross-reactivity and estimation of antibody titers against BeV and BjV were assessed by Ouchterlony double diffusion test. The results show that in pre-treatment protocol AE and BAv presented very similar effects (about 70% of inhibition for edematogenic and 40% for hemorrhagic activities). However, BAv poorly inhibited edema and hemorrhage in post-envenomation protocol, whilst, in contrast, AE was significantly active even when used after BeV injection. AE was able to inhibit all the tested enzymatic activities of BeV, while BAv was active only against hyaluronidase activity, which could justify the low effectiveness of BAv against BeV-induced local effects in vivo. Ouchterlony's test showed positive cross-reactivity against BeV, but the antibody titers were slightly higher against BjV. Together, these data indicate that despite the presence of immunological cross-reactivity, Brazilian polyvalent bothropic antivenom presented low inhibitory potential against biological and enzymatic effects of BeV, illustrating the need for new strategies in the production of antivenom with broad neutralizing potential in the treatment of Bothrops spp. envenomation throughout the country. Together, the results highlight the antiophidic potential of J. gossypiifolia, suggesting that it can be considered a potential adjuvant in the treatment of bothropic envenomation local effects.

Campylobacter hyointestinalis Isolated from Pigs Produces Multiple Variants of Biologically Active Cytolethal Distending Toxin.

Campylobacter hyointestinalis isolated from swine with proliferative enteritis often is considered to be pathogenic. While the precise virulence mechanisms of this species remain unclear, we have recently identified a cytolethal distending toxin (cdt) gene cluster in C. hyointestinalis isolated from a patient with diarrhea (W. Samosornsuk et al., J Med Microbiol, 27 July 2015, http://dx.doi.org/10.1099/jmm.0.000145). However, the sequences of the cdt genes in C. hyointestinalis were found to be significantly different and the gene products are immunologically distinct from those of other Campylobacter species. In this study, we demonstrate the presence of a second variant of the cdt gene cluster in C. hyointestinalis, designated cdt-II, while the former is named cdt-I. Sequencing of the cdt-II gene cluster and deduced amino acid sequences revealed that homologies between the subunits CdtA, CdtB, and CdtC of ChCDT-I and ChCDT-II are 25.0, 56.0, and 24.8%, respectively. Furthermore, the CdtB subunit of ChCDT-II was found to be immunologically unrelated to that of ChCDT-I by Ouchterlony double gel diffusion test. Recombinant ChCDT-II also induced cell distention and death of HeLa cells by blocking the cell cycle at G2/M phase. Interestingly, the cdt-II genes were detected in all 23 animal isolates and in 1 human isolate of C. hyointestinalis, and 21 of these strains carried both cdt-I and cdt-II gene clusters. Altogether, our results indicate that ChCDT-II is an important virulence factor of C. hyointestinalis in animals.

Occurrence of bacterial black shoot disease of European pear in Yamagata Prefecture

Black lesions on shoots of European pear trees observed in an orchard in Yamagata Prefecture in May 2007 were suspected to be caused by a bacterial pathogen. The surface of the colonies isolated on a high sucrose medium did not have the crater morphology that is characteristic of E. amylovora bvs. 1–3, and a specific DNA fragment was amplified from the isolates in the PCR using the EprpoD primer set. The partial sequences of the 16S rRNA gene placed the isolates in the genus Erwinia. The isolates differed serologically from E. amylovora biovars and E. pyrifoliae in an Ouchterlony double-diffusion test although their bacterial properties suggested that they are closely related to E. amylovora biovars and E. pyrifoliae. In a DNA–DNA hybridization test, the relatedness between the isolates and E. amylovora biovars or E. pyrifoliae did not exceed 70% level, indicating that they are independent species. Thus, the isolates belongs to the genus Erwnia but are not E. amylovra or E. pyrifoliae. After succulent pear shoots were injected with bacterial suspensions (109, 108, 107 and 106 cfu/ml) of the isolates, lesions formed with 109 and 108 cfu/ml, but the disease incidence with 108 cfu/ml was much lower than with E. amylovora and E. pyrifoliae. Virulence of the present isolates is thus thought to be very weak. On the basis of these results, we consider that this is a new shoot disease of European pear. In the 2007 season, all affected trees were pulled out after harvest. No symptoms have been observed in field surveys since the fruitlet season in 2007.

New Zealand grasslands revisited: identification ofCocksfoot mild mosaic virus

In the springs of 2003 and 2004, Bromus diandrus plants showing conspicuous mosaic symptoms were observed at Waitati, East Otago, New Zealand. Electron microscopic examination of sap samples in 2% phosphotungstate (pH 6.5) revealed large numbers of isometric particles 25 nm in diameter. The virus was mechanically transmitted to Setaria italica and Secale cereale cvv. Amilo and Rahu but not to Avena sativa, Chenopodium spp., Hordeum vulgare, Nicotiana benthamiana, Triticum aestivum or Zea spp. In Ouchterlony double diffusion tests, sap from infected plants and purified particles reacted strongly with antisera to Cocksfoot mild mosaic virus (CMMV and CMMV-Phleum mottle).Weaker reactions were observed with an antiserum to Panicum mosaic virus and there were no reactions with antisera to eight other sobemoviruses and ten other viruses known to infect the Poaceae. ELISA was used to test archived and fresh samples for CMMV and other viruses known to infect the Poaceae. CMMV was detected in B. diandrus and Ryegrass mosaic virus was detected in Holcus lanatus and Lolium perenne. However, Brome mosaic virus, Brome streak virus, Soilborne wheat mosaic virus, Wheat dwarf virus and Wheat streak mosaic virus were not detected in samples collected between 1995–2005 from Auckland and the South Island of New Zealand. This is the first report of CMMV in the Southern Hemisphere.

Genetic and immunological analysis of a novel variant of Shiga toxin 1 from bovine Escherichia coli strains and development of bead-ELISA to detect the variant toxin.

A novel variant of Shiga toxin 1 (Stx1) was identified from bovine Escherichia coli strains. The stx1 variant genes designated as stx1v51 and stx1v52 were cloned and sequenced. The two variant genes differed each other by 2 bp, but the deduced amino acid sequences of the two Stx1 variant toxins were the same and had 94% and 92% homology to that of prototype A and B subunits of Stx1, respectively. The variant toxin designated as Stx1v52 was purified to homogeneity. Although inhibition of protein synthesis in vitro by purified Stx1v52 was almost equal to that of purified Stx1, Vero cell cytotoxicity and mouse lethality of Stx1v52 were several folds lower than those of prototype Stx1. In Ouchterlony double gel diffusion test, the precipitin line between Stx1v52 and Stx1 formed a spur against anti-Stx1 serum but was fused against anti-Stx1v52 serum. Stx1v52 and Stx1v52-specific-bead-ELISA was developed, and both Stx1 and Stx1v52 could be detected with high sensitivity using Stx1v52 conjugate. However, Stx1v52 but not Stx1 could be detected with Stx1v52-specific bead-ELISA.

Serological Differences among Erwinia amylovora Biovars

Serological properties were compared among four biovars of Erwinia amylovora. Two biovars (bvs. 1 and 2) from Maloideae sources outside of Japan, one (bv. 3) from Rubus idaeus and one causing bacterial shoot blight of pear (bv. 4) had different reactions in Ouchterlony double diffusion tests using living cells and lipopolysaccharides (LPS) as antigens. These findings indicate that E. amylovora can be classified into three serotypes; i.e., bvs. 1 and 2, bv. 3 and bv. 4. From results of Ouchterlony double diffusion tests and immunoblots, the specific antigen of the three serotypes may each exist in the LPS of E. amylovora strains.

Fever, cough, and nodules on ankles

A 26-year-old woman with fever; headache, and a dry cough was admitted in September, 1998, to the Hyogo College of Medicine Hospital with multiple nodules on her chest radiograph. She had no relevant medical history and was in good general health. She had not travelled abroad. Her job was in an electrical appliance shop. Examination on admission showed cervical lymphadenopathy, leucocytosis 16·8×106/L, and eosinophilia (61%). There were no neurological abnormalities and her optic fundae were normal. Chest radiography and computed tomography (CT) showed multiple nodules in both lung fields. The size and location of these shadows changed during her stay in hospital. CT, magnetic resonance imaging (MRI), and ultrasonography of the liver were done. Non-contrast CT showed multiple low-density hepatic nodules. Hepatic-arterial-dominant phase-contrast CT showed several well-defined hepatic nodules. Portal-venous-dominant phase-contrast CT showed homogeneous enhancement of the lesions. MRI findings also suggested an inflammatory process, not a malignant tumour or haemangioma. Histological examination of specimens obtained from liver biopsy and transbronchial lung biopsy showed only extensive eosinophilic infiltration. There were elevated concentrations of immunoglobulin E (IgE, radioimmunosorbent test 8980 U/mL), specific IgE against Ascaris lumbricoides (radioallergosorbent test 74 UA/mL), and specific IgE against Anisakis simplex (radioallergosorbent test 1·81 UA/mL). Ouchterlony's double diffusion test showed a strong precipitin band against the larval excretory-secretory antigen of Toxocara canis. ELISA with the larval excretory-secretory antigen of T canis was positive. There was a small brown itchy nodule on her left ankle on admission. This was thought to be prurigo and was treated unsuccessfully with topical steroids. Similar skin lesions then appeared on the opposite leg.

Evidence of immunological Responses by a Host Fish (Ambloplites rupestris) and Two Non-Host Fishes (Cyprinus carpio and Carassius auratus) to Glochidia of a Freshwater Mussel (Villosa iris)

ABSTRACT Immunological responses of fishes to glochidia were evaluated using glochidia of the rainbow mussel (Villosa iris) to infest a host species, rock bass (Ambloplites rupestris), and two nonhost species, common carp (Cyprinus carpio) and goldfish (Carassius auratus). Ouchterlony double-diffusion tests showed that host and nonhost species expressed a humoral defense factor specific to glochidial antigens after induced infestation with glochidia. Precipitin bands were observed in tests on infested fishes but not in tests on uninfested fishes. Microagglutination tests showed that host and nonhost species that were uninfested, infested, or reinfested with glochidia all expressed some agglutination response to glochidial antigens. Experimental fishes had specific humoral defense factors that reacted immunologically to glochidia tissue.

Occurrence of Squash Mosaic Comovirus Infecting Summer Squash (Cucurbita pepo) in South Dakota and Transmission by Diabroticabarberi.

Summer squash (Cucurbita pepo L.) exhibiting strong mosaic symptoms and leaf deformations were found in Brookings County during the summers of 1994 and 1995. Four isolates were mechanically transmitted to C. pepo cv. Early Prolific Straightneck. Ouchterlony double diffusion tests (conducted both in Arkansas and South Dakota) and protein-A enzyme-linked immunosorbent assays (ELISAs) with squash mosaic virus (SqMV) antisera provided by R. C. Gergerich were positive for SqMV. Pumpkin mosaic virus, a strain of SqMV, was previously reported infecting pumpkin in South Dakota (1). The pumpkin strain was differentiated from other strains of SqMV by its ability to infect lentil (Lens esculenta Moench), garden pea (Pisum sativum L.), and fenugreek (Trigonella foenum-graecum L.). The South Dakota isolates in this study did not infect these hosts, although the Arkansas isolate was found to infect Pisum sativum cvs. Little Marvel and Dwarf Gray Sugar Pod when tested serologically. The presence of adult Diabrotica undecimpunctata howardii Barber and D. barberi (Smith and Lawrence) was noted on infected plants in the area. Transmission tests were conducted with D. barberi provided by the USDA-ARS Northern Grain Insect Research Laboratory, Brookings, SD. The beetles had been previously reared on artificial diet media and germinated corn seed. Individual beetles were given a 48-h acquisition access period on infected or healthy detached squash leaves in petri dishes followed by a 48-h inoculation access period on Early Prolific Straightneck squash plants (1 beetle per plant). Beetles were removed at the end of this period, and the plants were grown under greenhouse conditions for 3 weeks. Plants were assayed for SqMV by protein-A ELISA. Transmission percentages of SqMV were as follows: SqMV (Arkansas isolate), 16.7%; and four South Dakota isolates, 12.5, 11.4, 14.6, and 5.1%. Beetles fed on healthy plants did not transmit virus. The rate of transmission found in this study indicates that D. barbari serves as an efficient vector for the transmission of SqMV in field plantings. Reference: (1) W. N. Stoner. Proc. S. D. Acad. Sci. 56:178, 1977.

Detection of the arbuscular mycorrhizal fungus Scutellospora heterogama within roots using polyclonal antisera

The antigenic properties of the arbuscular mycorrhizal Scutellospora heterogama (BEG 35) were studied to develop a serological method for detecting the fungus in roots. Three polyclonal antisera were raised against the combined soluble and particulate fraction of spores of S. heterogama (BEG 35) and used in serological tests. When the soluble fraction of crushed spores was used as antigens in Ouchterlony double diffusion tests, all these antisera showed a high degree of specificity for the homologous fungus. No cross-reactivity was found with Glomus geosporum, Acaulospora spp., Entrophospora colombiana, Gigaspora spp. or other Scutellospora spp. Immunostaining of Western blots using one of the antisera was less specific but major cross-reactions were limited to fungi in Scutellospora, which all produced different banding patterns. This enabled immunostaining of Western blots to be used to detect and discriminate S. heterogama (BEG 35) and a morphologically similar Scutellospora sp. (BEG 40) in plant roots. There was no cross-reaction with uncolonized roots. All bands detected in extracts from colonized roots corresponded to bands detected in spore extracts, but some additional bands were unique to the spores. No significant reaction was observed with roots colonized by arbuscular mycorrhizal fungi outside the genus Scutellospora. The antiserum may thus be used to track species of Scutellospora within plant roots.

Purification and characterization of diamine oxidase (histaminase) from rat small intestine.

Diamine oxidase (DAO) was purified to homogeneity from rat small intestine, and its biochemical and immunochemical properties were studied. DAO was suggested to be a dimer of a 92 kDa subunit, and its isoelectric point was found to be 6.0. Histamine, putrescine, N tau-methylhistamine, and cadaverine were good substrates, with Km values ranging from 9.4 to 16.0 microM. Spermine and spermidine were not substrates. Both an immunoprecipitation study and Ouchterlony's double diffusion test involving antiserum against the purified DAO showed that the immunological properties of the DAOs from rat small intestine, thymus, and placenta were identical. Among small intestinal DAOs from different species, this antibody reacted to the guinea pig enzyme as strongly as to the rat enzyme, but the reaction was much weaker to the mouse enzyme than to the rat enzyme. The DAOs from rabbit and dog small intestine, pig kidney, and human placenta showed no reactivity toward this antibody.

Construction of mutant genes for a non-toxic verotoxin 2 variant (VT2vp1) of Escherichia coli and characterization of purified mutant toxins.

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1. Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed.

Purification, characterization and accumulation of three virus-induced cucumber peroxidases

Three anionic peroxidases (EC 1.11.1.7), named Prx1, 2, and 3, which are rapidly accumulated in cucumber (Cucumis sativus L., cv. Laura) reacting hypersensitively to tobacco necrosis virus, were purified to homogeneity. The three enzymes had an isoelectric point about 4.3, and the relative molecular masses of Prx1, 2, and 3 estimated by SDS-PAGE were 40 700, 38 000, and 37 100, respectively. These peroxidases had a similar pH stability, but differed in their specific activity, pH optimum, and thermal stability By Ouchterlony double diffusion tests with antisera raised against the three purified enzymes, close serological relationships have been demonstrated between the three peroxidases.

Purification and some properties of a Vero toxin 2 (Shiga-like toxin II) variant (SLT-IIva) of Escherichia coli isolated from infantile diarrhea

A variant of Vero toxin 2 (VT2), or Shiga-like toxin II (SLT-II) was purified from a cloned strain of Escherichia coli carrying a gene that encodes SLT-IIva, which is most closely related to SLT-IIv (VT2vp, VTe) in its nucleotide sequence. The purified SLT-IIva showed a slight difference from the purified SLT-IIv (VT2vp) in mobility on polyacrylamide gel disc electrophoresis and SDS-polyacrylamide gel slab electrophoresis and in pl, but both showed a similar potency in biological activities. Ouchterlony double diffusion test revealed that the purified SLT-IIva and SLT-IIv (VT2vp) formed a spur against anti-SLT-IIva and anti-SLT-IIv (VT2vp) antisera.

The two Staphylococcal bi-component toxins, leukocidin and gamma-hemolysin, share one component in common

Staphylococcal bi-component toxins, leukocidin and γ-hemolysin, consist of two protein components, i.e. F and S for leukocidin and HγI and HγII for γ-hemolysin. In this study we purified HγI and HγII to homogeneity from the culture medium of Staphylococcus aureus RIMD 310925 and compared their properties with those of F and S purified from the same source. The N-terminal 59- and C-terminal 2-residue amino acid sequences, apparent molecular mass, and isoelectri point of purified HγI were the same as those of F. In an Ouchterlony double diffusion test a fused line without spur was formed between F and HγI using either anti-F or anti-HγI antibodies. A synergistic action of F and HγII caused hemolysis of human red blood cells, and HγI acted synergistically with S to exhibit leukocidin activity. We conclude that the two toxins share one protein component (F = HγI) in common and leukocidin- and γ-hemolysin-specific activities are determined by S and HγII, respectively. It is also reported that the N-terminal 58-residue sequence of HγII is 72% similar to the corresponding sequence of S.

Characterization of antigens and allergens of date palm (Pheonix dactylifera) pollen. Immunologic assessment of atopic patients by whole extract and its fractions [corrected

Antigenic and allergenic components of date palm (Phoenix dactylifera) pollen were investigated to observe their effects on the skin test reactivity, lymphocyte blastogenesis and cytokine production in atopic and healthy individuals. Date pollen extracts were fractionated using SDS-PAGE and Sephacryl S-200 gel filtration. Western blotting of SDS-PAGE separated components with antiserum raised against whole pollen extract in rabbits revealed at least 22 immunoreactive bands ranging in molecular weight between 12 and 94 kD. The immunogenicity of the pollen extract was further confirmed by strong positive reactions in ELISA and Ouchterlony's double diffusion tests. Immunoblot analyses revealed IgG and IgE reactive components (14-94 kD for IgG and 12-90 kD for IgE) in the skin test-positive patients' sera against whole pollen extract. Fifteen of 60 atopics reacted positively to either whole or some fractions of date pollen extract when skin tested. In response to whole or components of date pollen extract atopic patients showed differential peripheral blood lymphocyte (PBL) proliferative response and cytokine (IL-2, IL-4) production when compared with PBL of normal subjects. Our findings strongly suggest that date palm pollen should be considered a reaginic component and should be included in the battery of allergens for determining the allergic status of atopic patients, particularly in those parts of the world where the date palm is grown commercially.

Distribution of multicatalytic proteinase in fish tissues

1. 1. MCP with similar immunoreactivity in Ouchterlony double-diffusion test and Western blot analysis was found in various white croaker tissues. 2. 2. The MCP content in different tissues was evaluated by immunoassay. The enzyme level was much higher in gonad than in any other organ tested, suggesting an important role of MCP in the process leading to embryonic development.

Purification, characterization and serological properties of two virus isolates associated with the maize stripe disease in Mauritius

Two virus isolates associated respectively with the maize stripe and the maize chlorotic stripe syndromes in Mauritius, denoted MSM and MCS, have been purified and characterized. A single nucleoprotein band of buoyant density 1.289 g/ml was recovered from both MSM and MCS extracts after isopycnic centrifugation in CsC1 gradients. Rate zonal centrifugation in 10–40% sucrose gradients of extracts of leaves infected by each isolate resulted in five light-scattering zones. The highest absorbency at 254 nm was associated with the third fraction from the top. Analysis of purified proteins by SDS-PAGE revealed a single capsid protein of Mr 34000 in preparations of each isolate. Large quantities of a non-capsid protein ofMr 23000 were extracted from both MSM- and MCS-infected material with a concentration of 1 75 and 2.35 mg/g of infected tissue, respectively. No such bands were detected in preparations from healthy leaves or leaves infected with maize streak virus (MSV). Both the 34000 and 23 000 proteins could be recovered from partially purified virus extracts. The property of the non-capsid protein to solubilize above and crystallize below pH 60 was used as the basis for a rapid diagnostic test. Electron microscopy of purified preparations of both isolates showed filamentous particles less than 5 nm in diameter and of different lengths. Several particle configurations, including circular, coiled and unfolded structures, were present, and were typical of a tenuivirus. Antisera raised in rabbits against the two nucleoproteins, each had titres of 1/32 both in homologous and reciprocal Ouchterlony double-diffusion tests. Sap extracts of both MSM- and MCS-infected plants gave strong reactions with antisera to maize stripe virus (MSpV) from Reunion, Kenya and the USA. The most sensitive diagnosis was achieved using F(ab′)2 ELISA. Both MSM and MCS should be considered isolates of maize stripe virus (MSpV). Although MSM and MCS induce different symptoms, they were indistinguishable in our tests; we propose that they are the same and that symptom differences arise as a result of late and early infection, respectively.

A simple purification method of Vibrio cholerae non-O1 hemagglutinin/protease by immunoaffinity column chromatography using a monoclonal antibody.

A new simple purification method (I) for Vibrio cholerae non-O1 hemagglutinin/protease (NAG-HA/P) was developed. The method (I) requires only an immunoaffinity column chromatography using a monoclonal antibody against NAG-HA/P. The method (I) is much simpler than previously reported purification method (II) (Honda, T. et al, Infection and Immunity 57: 2799-2803, 1989) which required four or more complicated chromatographic procedures. Method (I) also gave an improved recovery rate (about 27%) compared with (II). The molecular weight of NAG-HA/P purified by method (I) was mainly 34 kilodaltons (kDa) with a little of 32 kDa, whereas that of NAG-HA/P purified by (II) was usually 32 kDa. Immunological analysis by the Ouchterlony double gel diffusion test and Western blotting test using polyclonal antibody against 32 kDa protein revealed that the 34 and 32 kDa proteins are immunologically indistinguishable and thus it is supposed that 34 K protein is an isoform or a preform of the 32 K protein.

Evidence for the expression of morphological and biochemical characteristics of C3‐photosynthesis in chlorohyllous callus cultures of Zea mays

A Zea mays callus culture containing chlorophyll was established and grown photomixotrophically. Cell chloroplast structure, and pigment and soluble protein contents were examined. Expression of some key enzymes of C4 carbon metabolism was compared with that of etiolated (heterotrophic) and green photoautotrophic leaves. Chlorophyll content of the callus was 15–20% that of green leaves. Soluble protein content of callus was half that of leaf cells. Electron microscopic observations showed that green callus cells contained only typical granal chloroplasts. Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.38) activities in green callus were ca 30% those of green leaves but 2–3 times higher than in etiolated leaves. Quantitative enzyme protein determination, using antibodies specific to maize leaf Rubisco showed that the chloroplastic carboxylase represented about 7% of total soluble protein in green callus, in parallel to its low chlorophyll content. The specific activity of Rubisco in callus and leaves was unchanged.Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activity in green callus was about 20% that of green leaves and similar to that measured in etiolated leaves. Apparent Km (PEP) values (0.08 mM) for PEPC isolated from green callus and etiolated leaves were very different from values (0.5 mM) obtained with PEPC from green leaves. These kinetic characteristics together with the absence of inhibition by malate and activation by glucose‐6‐phosphate suggest that the properties of PEPC isolated from green callus and etiolated maize leaves are very similar to those of PEPPC from C3 plants. Using PEPC antibodies specific to green maize leaf enzyme, immunotitration of PEPC preparations containing identical enzyme units allowed complete precipitation of the green leaf enzyme with increasing antibody volumes. In contrast, 60–70% of the activity of PEPC from etiolated and green callus was inhibited, suggesting low affinity for the maize green leaf PEPC antiserum (typical C4 form). Ouchterlony double diffusion tests revealed only partial recognition of PEPC in green callus and etiolated leaves.NAD‐malate dehydrogenase (NAD‐MDH, EC 1.1.1.37) activity in callus was 2 and 3 times higher, respectively, than in etiolated and green leaves. NADP‐malic enzyme (NADP‐ME, EC 1.1.1.40) activity in callus cultures was much lower than in green leaves. All our data support the hypothesis that cultures of fully dedifferentiated chlorophyllous tissues of Zea mays possess a C3‐like metabolism.