Structure and distribution of lignin in primary and secondary cell walls of maize coleoptiles analyzed by chemical and immunological probes

Lignin is an integral constituent of the primary cell walls of the dark-grown maize (Zea mays L.) coleoptile, a juvenile organ that is still in the developmental state of rapid cell extension. Coleoptile lignin was characterized by (i) conversion to lignothiolglycolate derivative, (ii) isolation of polymeric fragments after alkaline hydrolysis, (iii) reactivity to antibodies against dehydrogenative polymers prepared from monolignols, and (iv) identification of thioacidolysis products typical of lignins. Substantial amounts of lignin could be solubilized from the coleoptile cell walls by mild alkali treatments. Thioacidolysis analyses of cell walls from coleoptiles and various mesocotyl tissues demonstrated the presence of guaiacyl-, syringyl- and (traces of)p-hydroxyphenyl units besidesp-coumaric and ferulic acids. There are tissue-specific differences in amount and composition of lignins from different parts of the maize seedling. Electron-microscopic immunogold labeling of epitopes recognized by a specific anti-guaiacyl/syringyl antibody demonstrated the presence of lignin in all cell walls of the 4-d-old coleoptile. The primary walls of parenchyma and epidermis were more weakly labeled than the secondary wall thickenings of tracheary elements. No label was found in middle lamellae and cell corners. Lignin epitopes appeared first in the tracheary elements on day 2 and in the parenchyma on day 3 after sowing. Incubation of coleoptile segments in H₂O₂ increased the amount of extractable lignin and the abundance of lignin epitopes in the parenchyma cell walls. Lignin deposition was temporally and spatially correlated with the appearance of epitopes for prolinerich proteins, but not for hydroxyproline-rich proteins, in the cell walls. The lignin content of coleoptiles was increased by irradiating the seedlings with white or farred light, correlated with the inhibition of elongation growth, while growth promotion by auxin had no effect. It is concluded that wall stiffness, and thus extension growth, of the coleoptile can be controlled by lignification of the primary cell walls. Primary-wall lignin may represent part of an extended polysaccharide-polyphenol network that limits the extensibility of the cell walls.Abbreviations G, S, H guaiacyl, syringyl andp-hydroxyphenyl constituents of lignin - HRGP hydroxyproline-rich glycoprotein - LTGA lignothioglycolic acid - PRP proline-rich protein Dedicated to Professor Benno Parthier on occasion of his 65th birthdayDeceased 7 November 1996     

Isolation and characterization of extra- and intra-cellular metal proteinases produced in the spawn-running process ofHypsizygus marmoreus

Isolation and characterization of extra-(PE-1) and intra-cellular (PE-2) metal proteinases produced during the spawn-running process ofHypsizygus marmoreus were carried out. These enzymes were the most active toward Hammarsten casein at pH 7.0 (PE-1) and pH 6.5–7.5 (PE-2). The molecular weight and pl value of PE-1 were 29,500, 8.8 and those of PE-2 were 21,500, 8.4. Km values against the synthetic peptide substrate Z-Gly-l-Leu-NH₂ were 0.9×10⁻³M (PE-1) and 1.2×10⁻³M (PE-2). PE-1 was strongly inhibited by phosphoramidon, whereas PE-2 was weakly inhibited. These enzymes are considered to play an important role in providing nitrogenous substrates during fruit-body formation.     

Characterization of zinc effect to inhibit osteoclast-like cell formation in mouse marrow culture: Interaction with dexamethasone

The inhibitory effect of zinc compounds on osteoclast-like cell formation in mouse marrow culture in vitro was characterized. The bone marrow cells were cultured for 7 days in -minimal essential medium containing a well-known bone resorbing agent, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or prostaglandin E2 (PGE2). Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of 1,25(OH)2D3 (10-8 M) or PGE2 (10-6 M) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were enhanced by the presence of dexamethasone (10-9 to 10-6 M). The dexamethasone (10-7 M)-enhanced osteoclast-like cell formation was not inhibited by the presence of zinc sulfate (10-6 M) or zinc-chelating dipeptide (-alanyl-L-histidinato zinc; 10-6 M), although the zinc compounds had an inhibitory effect on osteoclastic formation in the absence of the steroid. The effect of dexamethasone was not seen, when the steroid was added at the later stage of culture with bone-resorbing agents. In this case, the inhibitory effect of zinc compounds was clearly revealed. This effect of zinc compounds disappeared in the presence of Ca2+-chelating agent (0.5 mM EGTA). The present study suggests that zinc compounds have an inhibitory effect at the stage of differentiation of preosteoclastic cells in bone marrow cell culture system. (Mol Cell Biochem 166: 145-151, 1997)     

Shoot growth dynamics and size-dependent shoot fate of a clonal plant,Festuca rubra, in a mountain grassland

The relation of the within-season and between-season patterns of shoot growth were compared in a clonal grass with long-lived shoots,Festuca rubra, in a mown mountain grassland. The growth rate of shoot length from spring to summer in a year was almost constant for each shoot irrespective of spring shoot length each year. The annual shoot growth rate from spring to spring was negatively correlated with the shoot length in the first spring. Shoots of different length and age therefore tended to converge over time to a population of identical shoot size, suggesting an equalizing effect of growth pattern on size structure. Shoot size (shoot length and number of leaves) influenced the fates of shoots. Larger shoots showed an increased incidence of both flowering and formation of intravaginal daughter shoots and a decreased incidence of death in the subsequent time period. The fates of shoots were independent of their age. Although the negatively size-dependent springto-spring annual shoot growth rate acted to decrease shoot size variation, the remaining variation within the shoot population was still sufficient to generate different fates of shoots. These fates were not related to the previous life history of individual shoots. There was a significantly positive effect of the shoot size at initiation on its life expectancy. This was mainly attributable to the positively size-dependent survival rate of shoots in the early stage (<1 year old) of shoot life history. Later on (> 1 year old), shoot size had little effect on the survival rate of shoots. Once small young shoots have survived this early stage (< 1 year old) in life history, they can grow vigorously, little affected by competition regardless of shoot size, and converge to a stable size structure of shoots of similar size. Only shoot size in the early stage ( < 1 year old) of life history is important for the persistence of a shoot population.     

Nα-Fmoc-O,O-(dimethylphospho)-L-tyrosine fluoride: A convenient building block for the solid-phase synthesis of phosphotyrosyl peptides

Summary Fmoc-O,O-(dimethylphospho)-l-tyrosine was converted into stable Fmoc-O,O-(dimethylphospho)-L-tyrosine fluoride by means of (diethylamino) sulfur trifluoride or cyanuric fluoride. This building block was used for efficient coupling of phosphotyrosine to the adjacent sterically hindered amino acid Aib or Ac₆c in, model peptide sequences as well as for the synthesis of the ‘difficult’ phosphotyrosine peptide Stat91^695–708. The phosphate methyl groups were cleaved on solid phase after peptide assembly by means of trimethylsilyl iodide in MeCN. Aib, α-aminoisobutyric acid Ac₆c, 1-amino-cyclohexyl-l-carboxylic acid; BOP, benzotriazol-l-yl-oxy-tris(dimethylamino) phosphonium hexafluorophosphate, CIP, 2-chloro-l, 3-dimethylimidazolidium hexafluorophosphate, DAST, (diethylamino)sulfur trifluoride; DBU 1,8-diazabicyclo[5.4.0]undec-7-ene; DCM, dichloromethane; DIEA, drisopropylethylamine; DMA dimethylacetamide; Fmoc, 9-fluorenylmethoxycarbonyl; HATU,O-(7-azabenzotriazol-l-yl)-1.1,3,3-tetramethyluronium hexafluorophosphate; HOAt, I-hydroxy-7-azabenzotriazole; HOBt,N-hydroxybenzotriazole; HPLC, high-performance liquid chromatography; MBHA, 4-methylbenzhydrylamine; MeCN, acetonitrile; NMP,N-methyl-2-pyrrolidinone; NMR, nuerear magnetic resonance; PS, polystyrene; PyBroP, bromotris(pyrrolidino)phosphonium hexafluorophosphate; Rink amide MBHA-PS, 4-(2′,4′-dimethoxyphenyl-Fmoc-aminophenyl)-phenoxyacetamido-norleucyl-MBHA-PS; TFA, trifluoroacetic acid; TMSI, trimethylsilyl iodide; TPTU, 2-(2-pyridon-l-yl)-1,1,3,3-tetramethyluroniumfluoroborate; t_R, retention time; UNCA, arethane-protected amino acidN-carboxy anhydride Abbreviations for amino acids and nomenclature of, peptide structures follow the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature [Eur. J. Biochem., 138 (1984) 9].     

菊苣薄层培养花芽,营养芽分化中内源激素的动态变化

菊苣（Ｃｉｃｈｏｒｉｕｍ ｉｎｔｙｂｕｓＬ．）花梗薄层细胞培养于ＭＳ附加ＮＡＡ 和ＢＡ 或ＩＡＡ 和ＢＡ 的ＭＳ培养基上有花芽或营养芽分化． 花芽分化中内源ＩＡＡ、ＤＨＺ＋ ＤＨＺＲ、ｉＰＡ 含量明显增加,而Ｚ＋ ＺＲ变化不明显．营养芽分化中内源细胞分裂素含量增加明显,而ＩＡＡ 在培养前７ ｄ 含量下降,随后有所增加,在原基形成时含量达原初水平的２／３． 可见,花芽分化比营养芽分化所需内源ＩＡＡ／ＣＴＫ 比值要高     

Quantitative comparison of rat hepatocyte functions in two improved culture systems with or without rat liver epithelial cell line

We quantitatively evaluated two recently-developed novel techniques for hepatocyte cultivation in a dish level; that is, spheroid culture and membrane-supported collagen (CN) gel sandwich culture, in terms of cellular maintenance, albumin secretion and 7-ethoxycoumarin (7EC) metabolism to 7-hydroxycoumarin (7HC) as a marker for cytochrome P450 IA1 activity in the presence and absence of rat liver epithelial cell line (RLEC) during one month of culture, together with conventional coculture with RLEC in CN-coated dishes as a control. RLEC prevented spheroid loss caused by its detachment from the culture dishes often occurring in pure culture. CN-gel sandwich by itself improved remarkably hepatocyte maintenance when compared with CN-gel free systems, thereby resulting in enhancement of overall functional expressions as compared with CN-gel free systems. RLEC in CN-gel sandwhich, however, reduced cellular sustainment probably due to its suppression of hepatocyte growth. Although there were no significant differences in albumin secretion per cell among the five cultures examined, CN-gel sandwich expressed markedly higher 7EC metabolizing activity per cell, where RLEC presence had a preferable influence. Consequently, membrane-supported CN-gel sandwich was the most superior technique for hepatocyte cultivation from the standpont of both cellular maintenance and its functional expressions per cell.     

Degradation of bis(2-ethylhexyl) phthalate constituents under methanogenic conditions

The degradation of bis(2-ethylhexyl) phthalate (DEHP) and its intermediary hydrolysis products 2-ethylhexanol (2-EH) and mono(2-ethylhexyl) phthalate (MEHP) was investigated in a methanogenic phthalic acid ester-degrading enrichment culture at 37°C. 2-Ethylhexanoic acid (2-EHA), a plausible degradation product of 2-EH, was also studied. The culture readily degraded 2-EH via 2-EHA to methane which was formed in stoichiometric amounts assuming complete degradation of 2-EH to methane and carbon dioxide. MEHP was degraded to stoichiometric amounts of methane with phthalic acid as a transient intermediate. DEHP remained unaffected throughout the experimental period (330 days).Abbreviations 2-EH 2-ethylhexyl alcohol - 2-EHA 2-ethylhexanoic acid - BBP butylbenzyl phthalate - Be-CoA benzoyl Coenzyme A - CoA Coenzyme A - DEHP bis(2-ethylhexyl) phthalate - MEHP mono(2-ethylhexyl) phthalate - MSW municipal solid waste - PA phthalic acid - PAE phthalic acid ester - TMS trimethylsilyl derivative     

Ectopic lens induction in fish in response to the murine homeobox gene Six3

Recent findings show an unexpected conservation of genes involved in vertebrate and insect eye development. The Drosophila homeobox gene sine oculis is crucial for eye development. Its murine homologue, Six3 is expressed in the anterior neural plate, a region which is involved in lens induction in Xenopus. To examine whether Six3 participates in the process of eye formation, mouse Six3 was ectopically expressed in fish embryos. The results show that Six3 is sufficient to promote ectopic lens formation in the area of the otic vesicle and that retinal tissue is not a prerequisite for ectopic lens differentiation. Our findings suggest a conserved function for Six3 in metazoan eye development.     

Genetic mosaic analysis of decapentaplegic and wingless gene function in the Drosophila leg

 Genetically mosaic flies were constructed which lack a functional decapentaplegic (dpp) or wingless (wg) gene in portions of their leg epidermis, and the leg cuticle was examined for defects. Although dpp has previously been shown to be transcribed both ventrally and dorsally, virtually the only dpp-null clones that affect leg anatomy are those which reside dorsally. Conversely, wg-null clones only cause leg defects when they reside ventrally – a result that was expected, given that wg is only expressed ventrally. Both findings are consistent with models of leg development in which the future tip of the leg is specified by an interaction between dpp and wg at the center of the leg disc. Null clones can cause mirror-image cuticular duplications confined to individual leg segments. Double-ventral, mirror-image patterns are observed with dpp-null clones, and double-dorsal patterns with wg-null clones. Clones that are doubly mutant (null for both dpp and wg) manifest reduced frequencies for both types of duplications. Duplications can include cells from surrounding non-mutant territory. Such nonautonomy implies that both dpp and wg are involved in positional signaling, not merely in the maintenance of cellular identities. However, neither gene product appears to function as a morphogen for the entire leg disc, since the effects of each gene’s null clones are restricted to a discrete part of the circumference. Interestingly, the circumferential domains where dpp and wg are needed are complementary to one another. Received: 25 March 1996 / Accepted: 13 June 1996