Aquabacter spiritensis,gen. nov., sp. nov. an aerobic,gas-vacuolate aquatic bacterium

A gram-negative, rod-shaped, gas-vacuolate, aerobic, heterotrophic bacterium was isolated from Spirit Lake, Washington. It was further characterized by phase-contrast and electron microscopy and phenotypic tests. The isolate does not utilize carbohydrates. Deoxyribonucleic acid-ribosomal ribonucleic acid (rRNA) hybridizations showed that this bacterium is a member of rRNA superfamily IV where it occupies a separate position on the Azorhizobium caulinodans rRNA sub-branch which, is part of an rRNA cluster containing also the Beijerinckia, the Bradyrhizobium japonicum and the Rhodopseudomonas palustris sub-branches. Its relationship to other gas-vacuolated genera within rRNA superfamily IV is discussed. A new genus Aquabacter with one species Aquabacter spiritensis is proposed. The type strain is strain SPL-1 (=ATCC 43981=LMG 8611).     

Preparation of mycelial and yeast-like spheroplasts fromMucor rouxii by a lytic enzyme preparation fromPenicillium islandicum

Spheroplasts ofMucor rouxii were prepared from mycelial and yeast-like cells by use of aPenicillium islandicum enzymatic complex. This enzyme preparation, which presents high chitosanase and chitinase activities, was produced by growingP. islandicum either on mycelial or yeast-like walls ofM. rouxii. The presence of magnesium sulfate as an osmotic stabilizer was critical to obtain high yields of spheroplasts from mycelial forms. In the case of yeast-like cells, pretreatment with β-mercaptoethanol followed by magnesium sulfate was essential for extensive spheroplast production.     

Simultaneous Determination of 3,4-Dihydroxyphenylalanine, 5-Hydroxytryptophan, Dopamine, 4-Hydroxy-3-Methoxyphenylalanine, Norepinephrine, 3,4-Dihydroxyphenylacetic Acid, Homovanillic Acid, Serotonin, and 5-Hydroxyindoleacetic Acid in Rat Cerebrospinal Fluid and Brain by High-Performance Liquid Chromatography with Electrochemical Detection

A method using reversed-phase ion-pair high-performance liquid chromatography with electrochemical detection for the simultaneous determination of tryptophan (TRP), 3,4-dihydroxyphenylalanine (DOPA), and their metabolites in whole brain, small-brain parts, and cerebrospinal fluid of rats has been developed. The sample preparation requires only homogenization in perchloric acid and centrifugation before injection onto the column. With a LiChrosorb RP-18 (10 micrometer) column and a mobile phase consisting of a phosphate (NaH2PO4, 0.1 M)-methanol mixture with octylsulfonate (2.6 x 10(-3) M) at pH 3.35 and 26 degrees C, the separation of DOPA, dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 4-hydroxy-3-methoxyphenylalanine, TRP, 5-hydroxytryptophan (5-HTP), serotonin, and 5-hydroxyindoleacetic acid was achieved. The method has been applied to study the effect of alpha-monofluoromethyldopa alone and in combination with L-DOPA or L-5-HTP, on the catechol and 5-OH indole levels in brain and CSF of the rat.     

In vitro stimulation of mouse NK cell activity by phorbol esters: A pathway different from interferon-induced activation

Tumor promoters of the phorbol diester type were tested in vitro for their effect on mouse natural killer (NK) activity. The 12-O-tetradecanoyl-phorbol-13-acetate (TPA) significantly increased the spontaneous cytotoxicity of spleen cells preincubated with this reagent, with a maximal effect at 10–20 ng/ml. Higher concentrations of the weak tumor promoter phorbol-12, 13-dibenzoate (PDB) were required to display the same NK-enhancing property, while the inactive tumor promoter 4-αphorboldidecanoate was ineffective at any concentration. TPA-responding spleen cells displayed the same characteristics as classical NK cells: they were present in nude mice, did not adhere to nylon wool, bore the Thy-1 antigen, and displayed the same target cell specificity as interferon-activated NK cells. A major difference with the interferon-induced NK activation resides in the fact that the TPA-inducible increase in lytic activity does not require RNA and protein synthesis. Our results suggest two different NK activation pathways for IFN and phorbol esters.     

1′,2′-Dideacetylboronolide, an α-pyrone from Iboza riparia

The structural elucidation of 1′,2′-dideacetylboronolide, 5,6-dihydro-6-(3′-acetoxy-1′,2′-dihydroxyheptyl)2-pyrone, a new α-pyrone isolated from the leaves of Iboza riparia has been performed. Additionally, three sterols, sitosterol, stigmasterol and campesterol, have been identified in this species.     

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.     

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.