Further Daphniphyllum Alkaloids with Insecticidal Activity from the Bark of Daphniphyllum macropodum Miq.

Two new Daphniphyllum alkaloids, macropodumines J and K ( 1 and 2 , resp.), together with six known structurally related alkaloids, 3 – 8 , were isolated from the bark of Daphniphyllum macropodum Miq . The structures of the new compounds 1 and 2 were elucidated on the basis of a comprehensive analysis of their spectroscopic and chemical data. Macropodumine J ( 1 ) contains a CN group which is relatively rare in naturally occurring alkaloids. All isolated compounds were tested for their insecticidal activities against a number of insect species. Daphtenidine C ( 5 ) is the most active compound against Plutella xylostella. This is the first report of insecticidal properties of Daphniphyllum alkaloids.     

BRCA1 sequence variations in 160 individuals referred to a breast/ovarian family cancer clinic. Institut Curie Breast Cancer Group.

An account of familial aggregation in breast/ovarian cancer has become possible with the identification of BRCA1 germ-line mutations. We evaluated, for 249 individuals registered with the Institut Curie in Paris, the prior probability that an individual carried a mutation that predisposes to these diseases. We chose 160 women for BRCA1 analysis: 103 with a family history of breast cancer and 57 with a family history of breast-ovarian cancer. To detect small mutations, we generated and analyzed 35 overlapping genomic PCR products that cover the coding portion of the gene, by using denaturing gradient gel electrophoresis. Thirty-eight truncating mutations (32 frameshifts, 4 nonsense mutations, and 2 splice variants) were observed in 15% of women with a family history of breast cancer only and in 40% of those with a history of breast-ovarian cancer. Twelve of 25 distinct truncating mutations identified were novel and unique. Most BRCA1 mutations that had been reported more than five times in the Breast Cancer Information Core were present in our series. One mutation (5149del4) observed in two apparently unrelated families most likely originates from a common ancestor. The position of truncating mutations did not significantly affect the ratio of the risk of breast cancer to that of ovarian cancer. In addition, 15 DNA variants (14 missense mutations and 1 neutral mutation) were identified, 9 of which were novel. Indirect evidence suggests that seven of these mutations are deleterious.     

Attenuation of accumulation of neointimal lipid by pioglitazone in mice genetically deficient in insulin receptor substrate-2 and apolipoprotein E.

Rupture of vulnerable atherosclerotic plaques that are characterized by extensive neointimal accumulation of lipid is a cause of acute coronary syndromes. To identify whether insulin resistance alters atherogenesis, we characterized the composition of atherosclerotic lesions in the proximal aortas in mice deficient in apolipoprotein E (ApoE(-/-)) and in ApoE(-/-) mice in which insulin resistance was intensified by a concomitant heterozygous deficiency in insulin receptor substrate type 2 (IRS2(+/-) ApoE(-/-) mice). In addition, we characterized the effect of an insulin sensitizer, pioglitazone, on the atherogenesis in IRS2(+/-) ApoE(-/-) mice. The extent of the aortic intima occupied by lesion was increased in the IRS2(+/-) ApoE(-/-) compared with ApoE(-/-) mice (79 +/- 3% compared with 68 +/- 8%, p<0.05). Treatment with pioglitazone decreased the neointimal content of lipid in 20-week-old mice from 50 +/- 6% to 30 +/- 7%, p=0.005 and decreased the cellularity reflected by the multisection cross-sectional areas of lesions comprising cells in atheroma from 24 +/- 1% to 19 +/- 3%, p=0.018. Accordingly, genetically induced intensification of insulin resistance increases atheroma formation. Furthermore, attenuation of insulin resistance by treatment with pioglitazone decreases accumulation of lipid in the neointima.     

Epstein-Barr virus induces a unique pyrimidine deoxynucleoside kinase activity in superinfected and virus-producer B cell lines

Epstein-Barr (EB) virus induces a new pyrimidine deoxynucleoside kinase [thymidine kinase (dTk)] activity in Raji B lymphocyte cells after superinfection. This dTk activity is also present in small amounts in the HR-1 virus-producer cell line and in larger amounts in the B95-8 virus-producer line. The dTk activity induced by EB virus coelutes from DEAE-cellulose columns with deoxycytidine kinase (dCk) activity and elutes as a broad peak well separated from the large peaks of cellular dTk and dCk activities. This EB virus-induced pyrimidine deoxynucleoside kinase activity from HR-1 cells differs from cellular kinases in most basic biochemical properties but shares certain properties with the herpes simplex virus dTk.     

Drosophila P element-enhanced transfection in mammalian cells.

We constructed a gene transfer vector containing the herpes simplex virus type 1 thymidine kinase (TK) gene flanked by Drosophila P element terminal repeats (W. R. Engels, Annu. Rev. Genet. 17:315-344). This vector was introduced into mouse LTK- cells and enhanced the frequency of stable transformation to the TK+ phenotype by approximately 50-fold relative to a similar plasmid lacking the P element terminal repeats.     

A novel receptor‐like kinase involved in fungal pathogen defence in Arabidopsis thaliana

Plants are under constant attack from a variety of disease‐causing organisms. Lacking an adaptive immune system, plants repel pathogen attack via an array of pathogen recognition machinery. Receptor‐like kinases (RLKs) are involved in the recognition of pathogen‐associated molecular patterns (PAMPs) and activate resistance pathways against broad classes of pathogens. We have identified powdery mildew‐resistant kinase 1, an Arabidopsis gene encoding an RLK that is highly induced by chitin at early time points and localizes to the plasma membrane. Knockout mutants in pmrk1 are more susceptible to both Golovinomyces cichoracearum and Plectosphaerella cucumerina. Our data show that PMRK1 is essential in early stages of defence against fungi and provide evidence that PMRK1 may be unique to chitin‐induced signalling pathways. The results of this study indicate that PMRK1 is a critical component of plant innate immunity against fungal pathogens.     

High-irradiance responses induced by far-red light in grass seedlings of the wild type or overexpressing phytochrome A

The occurrence of phytochrome-mediated highirradiance responses (HIR), previously characterised largely in dicotyledonous plants, was investigated in Triticum aestivum L., Zea mays L., Lolium multiflorum Lam. and in both wild-type Oryza sativa L. and in transgenic plants overexpressing oat phytochrome A under the control of a 35S promoter. Coleoptile growth was promoted (maize, ryegrass) or inhibited (wild-type rice) by continuous far-red light (FRc). However, at equal fluences, hourly pulses of far-red light (FRp) were equally effective, indicating that the growth responses to FRc were not true HIR. In contrast, in maize and rice, FRc increased anthocyanin content in the coleoptile in a fluence-rate dependent manner. This response was a true HIR as FRp had reduced effects. In maize, anthocyanin levels were significantly higher under FRc than under continuous red light. In rice, overexpression of phytochrome A increased the inhibition of coleoptile growth and the levels of anthocyanin under FRc but not under FRp or under continuous red light. The effect of FRc was fluence-rate dependent. In light-grown rice, overexpression of phytochrome A reduced leaf-sheath length, impaired the response to supplementary far-red light, but did not affect the response to canopy shade-light. In grasses, typical HIR, i.e. fluence-rate dependent responses showing reciprocity failure, can be induced by FRc. Under FRc, overexpressed phytochrome A operates through this action mode in transgenic rice.Abbreviations FR far-red light - FRc continuous far-red light - FRp pulses of far-red light - HIR high-irradiance responses - LFR low-fluence responses - OPHYA transgenic rice overexpressing oat phytochrome A - Pfr far-red light-absorbing form of phytochrome - phyA phytochrome A - R red light - Rc continuous red light - VLFR very low-fluence responses - WT wildtype We thank Marcelo J. Yanovsky for his help with the photographs and Professor Rodolfo A. Sanchez for providing a reprint of the paper by P.J.A.L. de Lint. This work was supported by grants from UBA (AG041) and Fundacion Antorchas (A-13218/1-15) to J.J.C.     

Expression of functional oat phytochrome A in transgenic rice.

To investigate the biological functions of phytochromes in monocots, we generated, by electric discharge particle bombardment, transgenic rice (Oryza sativa cv Gulfmont) that constitutively expresses the oat phytochrome A apoprotein. The introduced 124-kD polypeptide bound chromophore and assembled into a red- and far-red-light-photoreversible chromoprotein with absorbance spectra indistinguishable from those of phytochrome purified from etiolated oats. Transgenic lines expressed up to 3 and 4 times more spectrophotometrically detectable phytochrome than wild-type plants in etiolated and green seedlings, respectively. Upon photo-conversion to the far-red-absorbing form of phytochrome, oat phytochrome A was degraded in etiolated seedlings with kinetics similar to those of endogenous rice phytochromes (half-life approximately 20 min). Although plants overexpressing phytochrome A were phenotypically indistinguishable from wild-type plants when grown under high-fluence white light, they were more sensitive as etiolated seedlings to light pulses that established very low phytochrome equilibria. This indicates that the introduced oat phytochrome A was biologically active. Thus, rice ectopically expressing PHY genes may offer a useful model to help understand the physiological functions of the various phytochrome isoforms in monocotyledonous plants.     

The interrelationships of the echinoderm classes: morphological and molecular evidence

The relationship between the five echinoderm classes has perplexed phylogeneticists for some time. Although each of the crinoids (C), asteroids (A), ophiuroids (O), echinoids (E) and holothuroids (H) are morphologically distinct, evidence from larval and adult morphology, molecular biology, and stratigraphy have failed to provide a single consensus solution. We have reviewed all available morphological and molecular data, added new data and reanalysed independent data sets individually and in combination, in order to resolve echinoderm class relationships. In total, we present 21 larval and 50 adult morphological characters, partial 28S-like large subunit rRNA gene data for 39 taxa and complete 18S-like small subunit rRNA gene data for 37 taxa. For a 5 taxon problem there are 105 possible rooted tree topologies, and yet we were consistently presented with three competing hypotheses when data sets were analysed both individually and in combination. The total evidence solution favoured (outgroup(C(A(O(E, H))))) although the alternative tree topology, (outgroup(C(O(A(E, H))))) was only one step longer and (outgroup(C((A, O),(E, H)))) was two steps longer. Only these three trees are serious contenders and the distribution of morphological characters suggests we should discount the solution placing ophiuroids as sister group to an asteroid+echinoid + holothurian clade. Thus we are left with (outgroup(C(A(O(E, H))))) and (outgroup(C((A, O),(E, H)))) as the two most plausible phylogenetic hypotheses. Our data showed high levels of phylogenetic signal and these trees best fit the available data.