Cytotoxic farnesyl glycosides from Pittosporum pancheri

Bioassay guided purification of the ethanolic extract of the bark of New Caledonian Pittosporum pancheri Brongn. and Gris (Pittosporaceae) led to the isolation and characterization of two new farnesyl monoglycosides, pancherins A and B. The structure of these compounds were determined on the basis of spectroscopic studies. The new compounds displayed a significant activity in the in vitro cytotoxic assay against KB cancer cell line, and pancherin A inhibits weakly farnesyl protein transferase.     

Steroidal saponins from Asparagus acutifolius

Six new steroidal saponins (1-6) were isolated from the roots of A. acutifolius L., together with a known spirostanol glycoside (7). Their structures were elucidated mainly by extensive spectroscopic analysis (1D and 2D NMR, FABMS and HRESIMS). Compounds 4-7 demonstrated antifungal activity against the human pathogenic yeasts C. albicans, C. glabrata and C. tropicalis with MICs values between 12.5 and 100 microg/ml.     

Parthenolide sensitizes cells to X-ray-induced cell killing through inhibition of NF-kappaB and split-dose repair

Human cancers have multiple alterations in cell signaling pathways that promote resistance to cytotoxic therapy such as X rays. Parthenolide is a sesquiterpene lactone that has been shown to inhibit several pro-survival cell signaling pathways, induce apoptosis, and enhance chemotherapy-induced cell killing. We investigated whether parthenolide would enhance X-ray-induced cell killing in radiation resistant, NF-kappaB-activated CGL1 cells. Treatment with 5 microM parthenolide for 48 to 72 h inhibited constitutive NF-kappaB binding and cell growth, reduced plating efficiency, and induced apoptosis through stabilization of p53 (TP53), induction of the pro-apoptosis protein BAX, and phosphorylation of BID. Parthenolide also enhanced radiation-induced cell killing, increasing the X-ray sensitivity of CGL1 cells by a dose modification factor of 1.6. Flow cytometry revealed that parthenolide reduced the percentage of X-ray-resistant S-phase cells due to induction of p21 waf1/cip1 (CDKN1A) and the onset of G1/S and G2/M blocks, but depletion of radioresistant S-phase cells does not explain the observed X-ray sensitization. Further studies demonstrated that the enhancement of X-ray-induced cell killing by parthenolide is due to inhibition of split-dose repair.     

Interest of RGD-containing linear or cyclic peptide targeted tetraphenylchlorin as novel photosensitizers for selective photodynamic activity

Destruction of the neovasculature is essential for tumor eradication by photodynamic therapy. Since the over-expression of integrins is correlated with tumor angiogenesis, we conjugated a photosensitizer (5-(4-carboxyphenyl)-10,15,20-triphenylchlorin or porphyrin) to the alpha(v)beta(3) integrin specific peptide RGD (H-Arg-Gly-Asp-OH) motif as a common sequence. We reported an efficient solid-phase synthesis of a new family of peptidic photosensitizers with linear or cyclic[RGDfK] RGD motif and compared conjugates in vitro selectivity and photodynamic activity. The conjugates were characterized by (1)H NMR, MALDI, UV-visible spectroscopy and singlet oxygen formation was performed. Chlorins containing linear and constrained RGD motif were incorporated up to 98- and 80-fold more, respectively, than the unconjugated photosensitizer over a 24-h exposure in human umbilical vein endothelial cells (HUVEC) over-expressing alpha(v)beta(3) integrin. Peptidic moiety also led to a non-specific increased cellular uptake by murine mammary carcinoma cells (EMT-6), lacking RGD binding receptors. Survival measurements demonstrated that HUVEC were greatly sensitive to conjugates-mediated photodynamic therapy.     

Temperature-mediated relationship between western flower thrips (Thysanoptera: Thripidae) and chrysanthemum

Modeling the effect of temperature on the sustainability of insect-plant interactions requires assessment of both insect and plant performance. We examined the effect of temperature on western flower thrips, Frankliniella occidentalis (Pergande), a generalist herbivore with a high reproductive rate, and chrysanthemum inflorescences, a high quality but relatively fixed, ephemeral resource for thrips population growth. We hypothesized that different thrips versus plant responses to temperature result in significant statistical interaction of temperature with thrips abundance and flower damage attributes over time. Experiments were conducted at five temperatures between 20.7 and 35.3 degrees C, with thrips infestation and time after infestation as main effects. Only minor, uncontrolled variations in relative humidity and light intensity may otherwise have influenced the results. High temperatures lead to an initially rapid increase in density of thrips followed by abrupt declines in abundance. The rate of floral senescence increased with temperature and thrips infestation, as indicated by a reduced fresh biomass and greater leaching of yellow pigments. Multiple regression indicated that indices of plant damage responded more directly to thrips density at low than high temperature, supporting the conclusion that temperature affected the outcome beyond what was predictable simply from differential plant and insect optima. The relative intensity of damage caused by individual thrips decreased with increasing temperature, likely caused by thrips competition and reduced survival, growth, and fecundity on depleted inflorescences. Reduced per capita damage at high temperature may be common in insects exploiting fixed plant resources that exhibit an accelerated rate of deterioration at high temperatures.     

Ca2+ channel currents and contraction in CaVbeta3-deficient ileum smooth muscle from mouse

Voltage activated L-type Ca(2+) channels are the principal Ca(2+) channels in intestinal smooth muscle cells. They comprise the ion conducting Ca(V)1 pore and the ancillary subunits alpha(2)delta and beta. Of the four Ca(V)beta subunits Ca(V)beta(3) is assumed to be the relevant Ca(V)beta protein in smooth muscle. In protein lysates isolated from mouse ileum longitudinal smooth muscle we could identify the Ca(V)1.2, Ca(V)alpha(2), Ca(V)beta(2) and Ca(V)beta(3) proteins, but not the Ca(V)beta(1) and Ca(V)beta(4) proteins. Protein levels of Ca(V)1.2, Ca(V)alpha(2) and Ca(V)beta(2) are not altered in ileum smooth muscle obtained from Ca(V)beta(3)-deficient mice indicating that there is no compensatory increase of the expression of these channel proteins. Neither the Ca(V)beta(2) nor the other Ca(V)beta proteins appear to substitute for the lacking Ca(V)beta(3). L-type Ca(2+) channel properties including current density, inactivation kinetics as well as Cd(2+)- and dihydropyridine sensitivity were identical in cells of both genotypes suggesting that they do not require the presence of a Ca(V)beta(3) protein. However, a key hallmark of the Ca(V)beta modulation of Ca(2+) current, the hyperpolarisation of channel activation is slightly but significantly reduced by 4 mV. In addition to L-type Ca(2+) currents T-type Ca(2+) currents could be recorded in the murine ileum smooth muscle cells, but T-type currents were not affected by the lack of Ca(V)beta(3). Both proteins, Ca(V)beta(2) and Ca(V)beta(3) are localized near the plasma membrane and the localization of Ca(V)beta(2) is not altered in Ca(V)beta(3) deficient cells. Spontaneous contractions and potassium and carbachol induced contractions are not significantly different between ileum longitudinal smooth muscle strips from mice of both genotypes. In summary the data show that in ileum smooth muscle cells, Ca(V)beta(3) has only subtle effects on L-type Ca(2+) currents, appears not to be required for spontaneous and potassium induced contraction but might have a function beyond being a Ca(2+) channel subunit.     

A simple genetic algorithm for the optimization of multidomain protein homology models driven by NMR residual dipolar coupling and small angle <Emphasis Type="Italic">X</Emphasis>-ray scattering data

Most proteins comprise several domains and/or participate in functional complexes. Owing to ongoing structural genomic projects, it is likely that it will soon be possible to predict, with reasonable accuracy, the conserved regions of most structural domains. Under these circumstances, it will be important to have methods, based on simple-to-acquire experimental data, that allow to build and refine structures of multi-domain proteins or of protein complexes from homology models of the individual domains/proteins. It has been recently shown that small angle X-ray scattering (SAXS) and NMR residual dipolar coupling (RDC) data can be combined to determine the architecture of such objects when the X-ray structures of the domains are known and can be considered as rigid objects. We developed a simple genetic algorithm to achieve the same goal, but by using homology models of the domains considered as deformable objects. We applied it to two model systems, an S1KH bi-domain of the NusA protein and the γS-crystallin protein. Despite its simplicity our algorithm is able to generate good solutions when driven by SAXS and RDC data.     

Experimental estrogen-induced hyperprolactinemia results in bone-related hearing loss in the guinea pig

Our group (Horner KC, Guieu R, Magnan J, Chays A, Cazals Y. Neuropsychopharmacology 26: 135-138, 2002) has earlier described hyperprolactinemia in some patients presenting inner ear dysfunction. However, in that study, it was not possible to determine whether hyperprolactinemia was a cause or an effect of the symptoms. To investigate the effect of hyperprolactinemia on inner ear function, we first developed a model of hyperprolactinemia in estrogen-primed Fischer 344 rats and then performed functional studies on pigmented guinea pigs. Hyperprolactinemia induced, after 2 mo, a hearing loss of approximately 30-40 dB across all frequencies, as indicated by the compound action potential audiogram. During the 3rd mo, the hearing loss continued to deteriorate. The threshold shifts were more substantial in males than in females. Observations under a dissection microscope revealed bone dysmorphology of the bulla and the cochlea. Light microscopy observations of cryostat sections confirmed bone-related pathology of the bony cochlear bulla and the cochlear wall and revealed morphopathology of the stria vascularis and spiral ligament. Scanning electron microscopy revealed loss of hair cells and stereocilia damage, in particular in the upper three cochlear turns and the two outermost hair cell rows. The data provide the first evidence of otic capsule and hair cell pathology associated with estrogen-induced prolonged hyperprolactinemia and suggest that conditions such as pregnancy, anti-psychotic drug treatment, aging, and/or stress might lead to similar ear dysfunctions.     

Inhibitory effect of a defensin gene from the Andean crop maca (Lepidium meyenii) against Phytophthora infestans

In this study, we report the isolation of a defensin gene, lm-def, isolated from the Andean crop 'maca' (Lepidium meyenii) with activity against the pathogen Phytophthora infestans responsible of late blight disease of the potato and tomato crops. The lm-def gene has been isolated by polymerase chain reaction (PCR) using degenerate primers corresponding to conserved regions of 13 plant defensin genes of the Brassicaceae family assuming that defensin genes are highly conserved among cruciferous species. The lm-def gene belongs to a small multigene family of at least 10 members possibly including pseudogenes as assessed by genomic hybridization and nucleotide sequence analyses. The deduced mature Lm-Def peptide is 51 amino acids in length and has 74-94% sequence identity with other plant defensins of the Brassicaceae family. The Lm-Def peptide was produced as a fusion protein using the pET-44a expression vector and purified using an immobilized metal ion affinity chromatography. The recombinant protein (NusA:Lm-Def) exhibited in vitro activity against P. infestans. The NusA:Lm-Def protein caused growth inhibition and hyphal damage at concentration not greater than 0.4 microM. In contrast, the NusA protein alone expressed and purified similarly did not show any activity against P. infestans. Therefore, these results indicate that the lm-def gene isolated from maca belong to the plant defensin family with activity against P. infestans. Its expression in potato, as a transgene, might help to control the late blight disease caused by P. infestans with the advantage of being of plant origin.