Pollen-pistil interactions in Populus: β-galactosidase activity associated with pollen-tube growth in the crosses P. nigra x P. nigra and P. nigra x P. alba

Summary In order to understand the nature of interspecific barriers in Populus, we have explored pollen/pistil interactions in intra- and interspecific crosses Populus nigra x P. nigra and P. nigra x P. alba. The kinetics of pollen-tube growth demonstrated that P. nigra and P. alba pollen tubes have distinct behavioral patterns inside P. nigra pistils. P. alba pollen tubes exhibit an unique S-shaped growth curve and an arrested growth site near the sylodium. P. nigra pollen tubes exhibit two growth phases, in the stigmatic tissues and in the ovarian cavity respectively. P. nigra and P. alba curves diverge 5 h after controlled pollination and could be related to a change in the physiology of the P. nigra pollen tube, which shifts from an autotrophic to a heterotrophic type of nutrition. Protein analysis of pollinated stigmatic extracts (0,6 and 20 h after pollination) revealed qualitative and quantitative differences that are related to the presence of either P. nigra or P. alba pollen tubes inside the stigmatic tissues. Increasing numbers of protein bands were detectable from 0 to 20 h after pollination only in intraspecific cross. Glycoproteins were detected, and the differences observed were dependent of the cross. -Galactosidase activity was found in pollinated stigmas, but an increase in its activity (one isozyme of pHi 4.2) between 6 h and 20 h after pollination was detected only in the intraspecific cross. This enzyme could play a role in heterotrophic pollen-tube nitrition, and its activity could be the final result of a series of interactions started by the initial pollen-stigma dialog.     

CD studies of peptides that mimic the four helicoidal sequences of the uteroglobin monomer

Summary Short peptides spanning the helicoidal sequences of the uteroglobin monomer (crystal forms P2₁ and C222₁) were synthesized and studied by circular dichroism spectroscopy. None of them showed any secondary structure in the absence of HFIP. However, most peptides achieved a helical conformation when this structuring agent was used, with the exception of the analogue corresponding to the helicoidal fragment 19–24 (helix II, crystal P2₁). These results indicate that other factors, such as interchain interactions, have to contribute to helix stabilization in the molecule. On the other hand, while peptides corresponding to N- and C-terminal fragments that contain the first and fourth helices of the monomer, respectively (1–14 and 48–70) achieved a -like structure when 10–15% of HFIP was used, this behaviour was not observed when TFE was used. Moreover, substitution of cysteine by -aminobutyric acid at position 3 increased both the helicity of fragment 1–14 and its ability to adopt a -like structure, but the opposite effect was observed for fragment 48–70 when -aminobutyric acid was introduced at position 69. These results indicate that this part of the protein might be sensitive to the chemical environment it is exposed to and that the two cysteine residues at positions 3 and 69 of the monomer could play a different role in the folding process.     

Poplar (Populus nigra L.) plants transformed with aBacillus thuringiensis toxin gene: insecticidal activity and genomic analysis

Insect-resistant poplar (Populus nigra L.) plants have been produced by infecting leaves withAgrobacterium tumefaciens strains carrying a binary vector containing different truncated forms of aBacillus thuringiensis (B.t.) toxin gene under a duplicated CaMV 35S promoter. Putative transgenic plants were propagated by cuttings at two experimental farms (in Beijing and Xinjiang, China). At 2–3 years after transformation, 17 of them were selected on the bases of insect-tolerance and good silvicultural traits, and evaluated for insect resistance, for the presence of theB.t. toxin DNA fragment (Southern blots and PCR) and for the expression of the transgene (western and northern blots). Somaclonal variation, as suggested by the appearance of permanent changes in the shape of the leaves, was also investigated with molecular tools (RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA) and microsatellite DNA).Bioassays withApochemia cineraius andLymantria dispar on the leaves of the selected clones showed different and, in some cases, high levels of insecticidal activity. The molecular analysis demonstrated integration and expression of the foreign gene. Somatic changes were correlated to extensive genomic changes and were quantified in dendrograms, in terms of genomic similarity. The analysis of control plants suggested that genomic changes were correlated to thein vitro culture step necessary forA. tumefaciens-mediated gene transfer, rather than to the integration of the foreign genes.Three transgenic clones (12, 153 and 192), selected for insect resistance, reduced morphological changes and promising silvicultural traits, are now under large-scale field evaluation in six different provinces in China.     

In situ detection of expression of thegus reporter gene in transgenic plants: ten years of blue genes

Among the methods now available to localize the sites of gene expression in plant materials, reporter genes based on thegus (uidA) gene ofEscherichia coli, which encodes a -glucuronidase (E.C. 3.2.1.31; GUS), have been the most widely used during the last ten years. The apparent simplicity of the histochemical GUS assay has been a major factor in the increase in articles usinggus genes. However, over the last four years, there have been occasional reports expressing doubts concerning the specificity of the observed localizations based on discrepancies between results obtained with GUS histochemistry and immunocytochemistry and/orin situ hybridization. This brief review compares the results obtained with immunocytochemistry with those obtained with various GUS substrates for histochemical studies. Certain sources of artefact are discussed, as are the limits that should be imposed on interpretation of GUS histochemistry results at the organ, tissue and cell levels.     

L－精氨酸和LDL对血管内皮细胞合成t－PA和PAI的影响

本实验采用酶联免疫分析法检测猪主动脉内皮细胞培养液中ｔＰＡ抗原、ｔＰＡ活性和ＰＡＩ活性,并观察了ＬＤＬ和Ｌ精氨酸对ｔＰＡ和ＰＡＩ的影响。结果表明：ＬＤＬ能明显降低ｔＰＡ抗原含量。同时伴有ｔＰＡ活性的降低和ＰＡＩ活性增高;Ｌ精氨酸能增加ｔＰＡ抗原含量伴ｔＰＡ活性增高,但对ＰＡＩ活性无明显影响。实验提示Ｌ精氨酸能保护血管内皮细胞,增加其ｔＰＡ合成,并能部分地拮抗ＬＤＬ降低ｔＰＡ合成的作用,可能对动脉粥样硬化早期防治有重要应用价值。     

Mature ferredoxin-NADP reductase with a glutaminyl residue at N-terminus from spinach chloroplasts

When 35%-acetone extract of spinach chloroplasts was separated by SDS-PAGE, ferredoxin-NADP reductase (FNR) appeared as a single band at a molecular mass of 35 kDa. After the polypeptides on the SDS-PAGE plate were electroblotted onto PVDF membrane, the FNR band was cut out and analyzed for N-terminal structure in a gas-phase protein sequencer. Two different FNR peptides were identified: one with glutamine at its N-terminus (Gln-FNR) and the other with -pyroglutamic acid (tFNR) fraction was extracted from chloroplasts with their loosely bound FNR (lFNR) fraction removed in advance. The tFNR fraction contained Gln-FNR only. The Gln-FNR could be highly purified by affinity chromatography using a ferredoxin column. The purified Gln-FNR was digested with arginyl endopeptidase for peptide mapping and partial sequence analysis. Primary structure of Gln-FNR differed from that of lFNR loosely bound FNR - tFNR tightly bound FNR - -pyroglutamic acid at N-terminus     

Fatty acid composition of vitellogenin from four teleost species

The fatty acid compositions of vitellogenin and liver from cod (Gadus morhua), rainbow trout (Oncorhynchus mykiss), turbot (Scophthalmus maximus) and wolffish (Anarhichas lupus) were determined. Vitellogenin was isolated from plasma of estradiol-17-treated fish by precipitation with EDTA-Mg²⁺ and distilled water or by high-performance ion-exchange chromatography. In all investigated species, vitellogenin contained 16–18% (w/w) lipid, in which polyunsaturated fatty acids, predominantly 20:5 (n-3) and 22:6 (n-3), comprised about 50% of the total fatty acids. The proportions of saturated, monounsaturated, polyunsaturated and (n-3) fatty acids in vitellogenin of the different species were generally similar, although the relative content of specific fatty acids was distinctive for each species. The distribution of fatty acids in total lipids of vitellogenin was highly consistent among individual females of each species. In contrast, liver fatty acid composition varied considerably, both within and between species. Altogether, the differences in the fatty acid composition of vitellogenin and liver from each species indicate that a specific selection of fatty acids occurs during the lipidation of vitellogenin.Abbreviations BHT butylated hydroxytoluene - E-17 estradiol-17 - EDTA ethylenedinitrilo tetra-acetic acid disodium salt dihydrate - FA fatty acids - FAME fatty acid methyl esters - HDL high density lipoproteins - PUFA polyunsaturated fatty acids - SD standard deviation - TLC thin-layer chromatography - VHDL very high density lipoproteins - VLDL very low density lipoproteins - v/v volume per volume - w/v weight per volume - w/w weight per weight     

In vitro investigation of α-amylase release from the digestive cells of the bivalve mollusc Pecten maximus: effect of second messengers and biogenic amines

The (neuro)endocrine control of enzyme release from invertebrate digestive cells remains poorly understood. A tissue dissociation procedure was developed to investigate the regulatory mechanisms of -amylase discharge from the cells of the stomach-digestive gland complex of the scallop Pecten maximus. The validity of the experimental system was tested by increasing the intracellular concentration of second messenger analogues (N ⁶,2-o-dibutyryl-adenosine-3,5 cyclic monophosphate and the ionophore A23187) known to mimic the activity of naturally occurring secretagogues in vertebrates: N ⁶,2-o-dibutyryl-adenosine-3,5 cyclic monophosphate increased the time and dose-dependent release of -amylase in a similar way as in vertebrates. A23187 was also very effective in inducing enzyme discharge. Since the in vitro bioassay was shown to be functional and because axon terminals were previously seen in close contact to -amylase secreting cells, the effect of some classic neurotransmitters was explored. Only the cholinergic agonist carbachol and dopamine evoked a secretory response. Maximal stimulation of -amylase release was reached at 10^-5 mol·l^-1 carbachol; at the same concentration dopamine was less effective than carbachol. By contrast, serotonin was totally inactive. The in vitro bioassay should prove useful for the identification of regulatory molecules involved in the control of enzyme discharge and to study stimulus secretion coupling mechanisms in scallop digestive cells.Abbreviations DBcAMP N ⁶, 2-O-dibutyryl-adenosine-3,5 cyclic monophosphate - cAMP adenosine-3,5 cyclic monophosphate     

Molecular phylogeny based on the κ-casein and cytochrome b sequences in the mammalian suborder Ruminantia

Nucleotide sequences for the -casein precursor proteins have been determined from the genomic DNAs or hair roots of the Ruminantia. The coding regions, exons 2, 3, and 4, were amplified separately via the three kinds of PCRs and then directly sequenced. The primers were designed from the sequence of bovine -casein gene; they were applicable for the amplification of the -casein genes from the 13 species in the Ruminantia except exon 2 of the lesser mouse deer. These results permitted an easy phylogenetic analysis based on the sequences of an autosomal gene. A phylogenetic tree was constructed from the mature K-casein sequences and compared with the tree of the cytochrome b genes which were sequenced from the same individuals. The Cervidae (sika deer, Cervus nippon) were separated from the branch of the Bovidae on the tree of -casein genes with a relatively high confidence level of the bootstrap analysis, but included in the branch of the Bovidae on the tree of cytochrome b genes. The -casein tree indicated a monophyly of the subfamily Caprinae, although the internal branches were uncertain in the Caprinae. The tree based on the nucleotide sequences of cytochrome b genes clearly showed the relationships of the closely related species in the genus Capricornis consisting of serow (C. smatorensis), Japanese serow (C. crispus), and Formosan serow (C. swinhoei). These results would be explained by the difference of resolving power between the -casein and the cytochrome b sequences. Correspondence to: K. Chikuni     

Presence and abundance of CENP-B box sequences in great ape subsets of primate-specific α-satellite DNA

CENP-B, a highly conserved centromere-associated protein, binds to -satellite DNA, the centromeric satellite of primate chromosomes, at a 17-bp sequence, the CENP-B box. By fluorescence in situ hybridization (FISH) with an oligomer specific for the CENP-B box sequence, we have demonstrated the abundance of CENP-B boxes on all chromosomes (except the Y) of humans, chimpanzee, pygmy chimpanzee, gorilla, and orangutan. This sequence motif was not detected in the genomes of other primates, including gibbons, Old and New World monkeys, and prosimians. Our results indicate that the CENP-B box containing subtype of -satellite DNA may have emerged recently in the evolution of the large-bodied hominoids, after divergence of the phylogenetic lines leading to gibbons and apes; the box is thus on the order of 15–25 million years of age. The rapid process of dispersal and fixation of the CENP-B box sequence throughout the human and great ape genomes is thought to be a consequence of concerted evolution of -satellite subsets on both homologous and nonhomologous chromosomes.Correspondence to: T. Haaf