Establishment and characterization of photosynthetic hairy root cultures of Datura stramonium

Twelve different lines of Datura stramonium (normal and hairy) root cultures were subjected to conditions which induce photoautotrophy. Two of the hairy root lines responded to induction, showing clearly a diminished growth rate when compared to heterotrophic cultures, an increase in chlorophyll, a net O₂ evolution, CO₂ fixation, and de novo synthesis of the ribulose 1,5 biphosphate carboxylase enzyme. A time course of growth and tropane alkaloid levels in the tissue and medium, revealed a correlation between the development of the photosynthetic apparatus and the increase in scopolamine. Although normal cultures did not grow photosynthetically, they showed some greening response under the first step of the induction. The correlation between development of photosynthesis and increase in scopolamine synthesis were corroborated with normal root cultures. This experimental model is used for the basic study of the regulatory enzymes involved in the biosynthesis of tropane alkaloids, as well as for the study of their mechanisms of transport.     

Gene transfer to inflorescence and flower meristems using ballistic micro-targeting

Direct gene transfer to floral meristems could contribute to cell-fate mapping, to the study of flower-specific genes and promoters, and to the production of transgenic gametes via the transformation of sporogenic tissues. Despite the wide potential of its applications, direct gene transfer to floral meristems has not been achieved so far because of the lack of suitable technology. We show in this paper that ballistic micro-targeting is the technique of choice for this purpose, and in this way, we were able to transfer genes efficiently into excised wheat immature spikes. Particle size was adjusted for optimal penetration into the L1 and L2 cell layers of the spikes with limited cell damage. Spikes at different developmental stages were shot either with a plasmid containing two genes involved in anthocyanin biosynthesis or with a plasmid bearing the uidA (-glucuronidase) gene. The transient expression of these marker genes was observed in the different developmental stages tested and in cells of both the L1 and the L2 layers. The transient expression of the uidA gene was significantly increased when the sucrose concentration in the culture medium was increased from 0.06 to 0.52 M. At the highest concentration, 100% of the targeted spikes expressed the uidA gene, with an average of 69 blue cells per spike. Twelve days after microtargeting, multicellular sectors showing transgene expression and containing up to 17 cells were found in 85% of the shot immature inflorescences. This indicated that targeted cells survived particle bombardment. Sectors were found in primordia of both vegetative and reproductive organs.     

Analysis of peptides from known proteins: Clusterization in sequence space

A combinatorial sequence space (CSS) model was introduced to represent sequences as a set of overlapping k-tuples of some fixed length which correspond to points in the CSS. The aim was to analyze clusterization of protein sequences in the CSS and to test various hypotheses about the possible evolutionary basis of this clusterization. The authors developed an easy-to-use technique which can reveal and analyze such a clusterization in a multidimensional CSS. Application of the technique led to an unexpectedly high clusterization of points in the CSS corresponding to k-tuples from known proteins. The clusterization could not be inferred from nonuniform amino acid frequencies or be explained by the influence of homologous data. None of the tested possible evolutionary and structural factors could explain the clusterization observed either. It looked as if certain protein sequence variations occurred and were fixed in the early course of evolution. Subsequent evolution (predominantly neutral) allowed only a limited number of changes and permitted new variants which led to preservation of certain k-tuples during the course of evolution. This was consistent with the theory of exon shuffling and protein block structure evolution. Possible applications of sequence space features found were also discussed.Correspondence to: H.A. Lim     

Identification of a Cholinergic-Specific Antigen Chol-1 as a Ganglioside

Abstract: An antiserum specific for cholinergic terminals was used to identify an antigen conserved between Elasmobranchs and mammals. Immunohistochemistry and a cytotoxicity test were used to assay the binding of antibody to mammalian terminals. Torpedo electric organ gangliosides totally abolished antibody binding. The highest inhibitory activity was associated with a single polysialoganglioside band on TLC plates. Neuraminidase altered the migration of the inhibitory activity on TLC plates. Antibody binding was inhibited by ganglioside fractions derived from chicken and mammalian brains. A summary of those tissues in which the antigen has been detected is presented. The possible function of the antigen is discussed.     

Application of adsorption isotherms to chromium adsorption onZ.ramigera

Summary Adsorption isotherms for the adsorption of chromium onZoogloea ramigera are developed. The rates were affected by the pH and temperature of adsorption medium. The biomass ofZ. ramigera at pH 2.0 where the optimum pH for biosorption lies exhibited the highest chromium adsorptive uptake capacity. In general, higher adsorptive uptake was observed at 25°C than 35°C and 45°C.     

Effect of alpha-tocopherol on the concentration of alpha-tocopherylquinone in human blood lipids

The electron-spin resonance (ESR) was applied to examine human plasma and red cell lipids. In the lipids, the signal of ESR of alpha-tocopherylquinone was recorded. The concentration of the latter in the lipids was found to be elevated in coronary heart disease. Intramuscular injection of 30% oily solution of alpha-tocopherylquinone in a dose of 2 ml two times a day over three days entailed an increase in the concentration of alpha-tocopherylquinone in plasma and red cell lipids.     

Proteins of the Brain Extracellular Fluid: Evidence for Release of S-100 Protein

Abstract: Extracellular protein fractions were obtained (1) by mild, isotonic irrigation of freshly perfused brain tissue; (2) by collection of proteins released into super-fusing medium by physiologically viable slices of rat hippocampus; and (3) by sampling the CSF of anesthetized rats. Analysis of the S-100 protein content of these fractions gave values of 2.8, 4.2, and 1.8 μg S-100/mg protein, respectively. These values were three- to sixfold higher than the S-100 content of the soluble cytoplasmic protein fractions from the same tissue. This several-fold higher S-100 content of the extracellular protein fractions relative to the intracellular cytoplasmic protein fractions indicates that S-100 is selectively released into the extracellular spaces of the brain. We suggest that the biological function of this CNS protein may involve intercellular transfer.