Postillumination CO2 fixation by wheat leaves

Postillumination CO₂ fixation by wheat leaves was studied following light-limited photosynthetic conditions. Dark CO₂ fixation showed two phases differing by their rates of CO₂ uptake and carbon metabolism. These two phases are related to preillumination light flux density. During the first 30s of darkness, assimilated CO₂ was found in PGA, alanine, malate and aspartate. After 5 min of darkness, it was additionally found in phosphorylated sugars.The lack of labelling of glycolate pathway intermediates shows that the Calvin cycle cannot run in the dark.The synthesized compounds indicate that reducing power but not ATP is available after turning the light off. This observation suggests that during pre-illumination, when light strictly limits photosynthesis, ATP supply would be the first limiting factor.

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Résumé La fixation post-illuminatoire de CO₂ par des feuilles de blé est étudiée, après une période de photosynthèse en lumière limitante.Les cinétiques de la vitesse de fixation du CO₂ après extinction présentent deux phases, se différenciant par la vitesse de fixation du CO₂ et par les voies métaboliques suivies par le carbone.Pendant les premières 30s d'obscurité, le CO₂ fixé est retrouvé principalement dans le PGA, l'alanine, le malate et l'aspartate. Après 5 min d'obscurité le carbone est retrouvé également dans les esters phosphorylés des oses.L'absence de radioactivité dans les intermédiaires de la voie du glycolate indique que le cycle de Calvin ne peut pas fonctionner á l'obscurité.Les composés synthétisés à l'obscurité suggèrent que du pouvoir réducteur est disponible. Par contre l'ATP ne l'est pas. Ainsi, durant la période de pré-illumination oú la lumière était strictement limitante la disponibilité en ATP serait le premier facteur limitant l'assimilation du CO₂.

     

Functional analysis of DM64, an antimyotoxic protein with immunoglobulin-like structure from Didelphis marsupialis serum.

Bothrops snake venoms are known to induce local tissue damage such as hemorrhage and myonecrosis. The opossum Didelphis marsupialis is resistant to these snake venoms and has natural venom inhibitors in its plasma. The aim of this work was to clone and study the chemical, physicochemical and biological properties of DM64, an antimyotoxic protein from opossum serum. DM64 is an acidic protein showing 15% glycosylation and with a molecular mass of 63 659 Da when analysed by MALDI-TOF MS. It was cloned and the amino acid sequence was found to be homologous to DM43, a metalloproteinase inhibitor from D. marsupialis serum, and to human alpha1B-glycoprotein, indicating the presence of five immunoglobulin-like domains. DM64 neutralized both the in vivo myotoxicity and the in vitro cytotoxicity of myotoxins I (mt-I/Asp49) and II (mt-II/Lys49) from Bothrops asper venom. The inhibitor formed noncovalent complexes with both toxins, but did not inhibit the PLA2 activity of mt-I. Accordingly, DM64 did not neutralize the anticoagulant effect of mt-I nor its intracerebroventricular lethality, effects that depend on its enzymatic activity, and which demonstrate the dissociation between the catalytic and toxic activities of this Asp49 myotoxic PLA2. Furthermore, despite its similarity with metalloproteinase inhibitors, DM64 presented no antihemorrhagic activity against Bothrops jararaca or Bothrops asper crude venoms, and did not inhibit the fibrinogenolytic activity of jararhagin or bothrolysin. This is the first report of a myotoxin inhibitor with an immunoglobulin-like structure isolated and characterized from animal blood.     

Engineering of a Single-Chain Variable-Fragment (scFv) Antibody Specific for the Stolbur Phytoplasma (Mollicute) and Its Expression in Escherichia coli and Tobacco Plants

From a hybridoma cell line (2A10) producing an immunoglobulin G1 directed against the major membrane protein of the stolbur phytoplasma, we have engineered scFv (single-chain variable-fragment) antibodies from the variable heavy (VH) and light (VL) domains of the immunoglobulin. The scFv gene was cloned and expressed in Escherichia coli. The expressed protein of 30 kDa could be recovered from the periplasmic fraction of the bacterial cells and was shown to be fully functional toward its phytoplasmal antigen, since enzyme-linked immunosorbent assay or immunofluorescence (IF) detection of the stolbur phytoplasma antigen by the scFv was identical to that of the native immunoglobulin. The scFv gene was then cloned in plasmid pBG-dAb-BIN of Agrobacterium tumefaciens to transform tobacco plants. The transformed plants were screened by PCR and Northern blotting for the presence and expression of the transgene, respectively, and by IF for expression of the scFv. One transgenic tobacco line, 1A6, was selected for challenge inoculation with the stolbur phytoplasma. When grafted on a stolbur phytoplasma-infected tobacco rootstock, the transgenic tobacco shoots grew free of symptoms and flowered after 2 months, while normal tobacco shoots showed severe stolbur symptoms during the same period and eventually died.     

A potent inhibitor of platelet activating factor from the saliva of the leech Hirudo medicinalis.

Leech saliva is shown to contain protein platelet aggregation inhibitors and a range of selective low molecular weight (LMW) aggregation inhibitors. Gel filtration on Bio-Gel P-2 (cut-off kDa) yields a protein fraction (Fr. I) and three LMW fractions. Fr. I inhibits aggregation induced by collagen, ADP, epinephrine and arachidonic acid. Of all the fractions, only one, Fr. II (LMW) specifically inhibits aggregation induced by platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine). Fr. II also inhibits thrombin-induced platelet aggregation. Fr. III inhibits aggregation induced by ADP, epinephrine and arachidonic acid, and Fr. IV only that induced by arachidonic acid. Fr. II also inhibits PAF- and thrombin-induced thromboxane generation in platelets, but does not inhibit arachidonic acid-induced thromboxane generation. Efforts to separate the anti-PAF from the anti-thrombin activity have been unsuccessful. The inhibition may therefore be due to a single inhibitor, though it may also be due to several inhibitors. Fr. II also inhibits superoxide anion production in formyl Met-Leu-Phe (fMLP)- and ionophore 23187- stimulated neutrophils. This may be due to the inhibition of the effects of PAF generated within the cell. Preliminary results suggest that the Fr. II inhibitor(s) is (are) amphipathic. The interaction of platelets with PAF and their interaction with the inhibitor(s) are mutually exclusive, and the inhibition may be competitive.     

Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens

Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.     

Linkage disequilibrium between phenylketonuria and RFLP haplotype 1 at the phenylalanine hydroxylase locus in Portugal

Summary RFLPs of 36 normal and 41 mutant alleles at the phenylalanine hydroxylase locus were determined in 31 Portuguese kindreds. A total of 14 haplotypes including 10 normal and 7 mutant alleles were observed. Almost 75% of all mutant alleles were confined within only two haplotypes, namely haplotype 9 (17.1%) and haplotype 1 (56.1%). This frequency of mutant haplotype 1 in Portugal is, to our knowledge, the highest for this mutant haplotype in all studies reported to date. Other mutant haplotypes were either rare (haplotype 2, 9.7%) or totally absent (haplotype 3, 0%). Only 24.5% of all mutant alleles were found to consistently carry identified mutations, particularly R261Q (9.8%), R252W (3.3%), R408W (1.6%) and I94 (3.3%). A new mutation, L249F, located in the seventh exon of the gene, accounted for 6.5% of all mutant alleles in our series. Interestingly, this mutant genotype was consistently associated with mutant haplotype 1 (P<0.01), as also observed for the R261Q mutation. It appears, therefore, that mutant haplotype 1 is genotypically heterogeneous in Portugal and that more than two mutations account for its prevalence in this country.     

Specific binding of placental acidic isoferritin to cells of the T-cell line HD-MAR

Comparative analysis of nuclear Overhauser effects show that the time average conformation of the wild-type and mutant Pro → Ala-35 Rhodobacter capsulatus cytochrome c2 are indistinguishable. The ring resonances of Phe-51 and Tyr-53 show that their ring flip rates increase in P35A. NH proton exchange studies show that the exchange rates of the NH of Gly-34 and the NπH of His-17 increase by ≈ 102 in P35A suggesting that their respective hydrogen bonds are destabilized in this protein. However, 3NH. 1H and 15N chemical shift data argue that these bonds are intact. These data are compatible if the replacement of a Pro with an Ala residue forms a cavity or increases local flexibility thus reducing steric hinderance and increasing solvent accessibility.     

Methionine biosynthesis in Saccharomyces cerevisiae: mutations at the regulatory locus ETH2. I. Genetic data

Summary Evidence has been obtained that sodium azide is an inhibitor of cell division in wild-type and aziA strains of Salmonella typhimurium. The bacteria grown in media containing sodium azide and glucose formed long filaments. It has been found that sodium azide had a stronger inhibitory effect on DNA synthesis than on cell mass increase. When filaments produced by azide action were transferred to azide-free medium very rapid increase in DNA content was observed during the first 45 min. After this time, when relative DNA content was increased the rate of DNA synthesis was reduced and cell divisions reappeared.Inhibitory effect of azide on DNA biosynthesis in vitro was observed with toluenized cells of S. typhimurium. Only ATP-dependent radioactive dTMP incorporation into DNA was affected by sodium azide. It had no effect on the incorporation in the absence of ATP.Mutant aziC was isolated in S. typhimurium by scoring for clones with normal cell division in the presence of sodium azide. Azide had much less effect on DNA biosynthesis in vivo and in vitro in aziC cells as compared with isogenic controls.This work was supported by the Polish Academy of Sciences within the project 09.3.1., and by the U.S. Public Health Service, grant No. 05-032-1.     

Ultrastructural modifications of frog urinary bladder epithelium under the influence of hypertonic media

Summary The frog urinary bladder undergoes a marked increase in its water permeability when incubated in hypertonic media. Many similarities are found between this effect and the hydrosmotic action of antidiuretic hormone. The ultrastructural modifications of the epithelium observed under the influence of serosal hypertonicity (the intercellular spaces are dilated while the tight junctions remain closed) lead us to assume that the pathways of water movement across the epithelium could be the same in this case and in hydrosmotic response to the hormone. In contrast, when the mucosal medium is made hypertonic, the ultrastructure is differently altered: the intercellular spaces are closed, the tight junctions show small vesicles and numerous large vacuoles appearing in the cytoplasm.