Insecticidal Constituents from the Argentine Liverwort Plagiochila bursata

The new 2,3‐secoaromadendrane 1 , together with the known compounds plagiochilines A and M ( 2 and 3 , resp.), fusicogigantone A ( 4 ), and 1,4‐dimethylazulene ( 5 ) were isolated from an Argentine collection of the liverwort Plagiochila bursata. Structures were elucidated by extensive 1D‐ and 2D‐NMR studies. Compounds 2 and 4 , incorporated to the larval diet at 100 μg per g of diet, reduced the larval growth of Spodoptera frugiperda (Lepidoptera: Noctuidae) by 66±29% and 25±8% and produced 55 and 75% larval mortality at early instars and 20 and 25% pupal mortality, respectively. Treatment with compound 2 also produced abdomen and wing malformation in adults leading to impossibility to mate.     

TGFβ induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

Background and aims: Transforming growth factor-beta (TGFβ) is known to potently inhibit cell growth. Loss of responsiveness to TGFβ inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGFβ and HB-EGF signal transduction via ADAM activation.Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGFβ. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGFβ was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGFβ was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown.Result: TGFβ-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGFβ induced shedding of proHB-EGF allowing HB-EGF-CTF to translocate to the nucleus. ADAM inhibitors blocked this nuclear translocation. TGFβ enhanced gastric cancer cell growth and ADAM inhibitors suppressed this effect. EGFR phosphorylation, HB-EGF-CTF nuclear translocation, and cell growth were suppressed in ADAM17 knockdown cells.Conclusion: HB-EGF-CTF nuclear translocation and EGFR transactivation from proHB-EGF shedding mediated by ADAM17 activated by TGFβ might be an important pathway of gastric cancer cell proliferation by TGFβ.     

Confocal microscopic observation of fusion between baculovirus budded virus envelopes and single giant unilamellar vesicles

We assayed fusion events between giant unilamellar vesicles (GUVs) and budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus), the envelopes of which have been labeled with the fluorescent dye Alexa Fluor 488. This involves observing the intensity of fluorescence emitted from the lipid bilayer of single GUVs after fusion using laser scanning microscopy. Using this assay system, we found that fusion between single GUVs and BV envelopes was significantly enhanced at around pH 5.0-6.0, which suggests that: (1) envelope glycoprotein GP64-mediated membrane fusion within the endosome of insect cells was reproduced in our artificial system; (2) acidic phospholipids in GUVs are necessary for this fusion, which are in agreement with the previous results with conventional small liposomes including large unilamellar vesicles and multilamellar vesicles; and (3) the efficiency of fusion is significantly affected by membrane properties that can be modulated by adding cholesterol to GUV lipid bilayers. In addition, the microscopic observation of BV-fused single GUVs showed that a weak interaction occurred between BVs and GUVs containing dioleoylphosphatidylserine at pH 6.0-6.5, and components of BV envelopes were unevenly distributed upon fusion with GUVs containing saturated phospholipid with cholesterol. We further demonstrated that when the recombinant membrane protein, adrenergic β₂ receptor, was expressed on recombinant BV envelopes, the protein distribution on BV-fused GUVs was also affected by their lipid contents.     

Regulation of the gibberellin pathway by auxin and DELLA proteins

The synthesis and deactivation of bioactive gibberellins (GA) are regulated by auxin and by GA signalling. The effect of GA on its own pathway is mediated by DELLA proteins. Like auxin, the DELLAs promote GA synthesis and inhibit its deactivation. Here, we investigate the relationships between auxin and DELLA regulation of the GA pathway in stems, using a pea double mutant that is deficient in DELLA proteins. In general terms our results demonstrate that auxin and DELLAs independently regulate the GA pathway, contrary to some previous suggestions. The extent to which DELLA regulation was able to counteract the effects of auxin regulation varied from gene to gene. For Mendel’s LE gene (PsGA3ox1) no counteraction was observed. However, for another synthesis gene, a GA 20-oxidase, the effect of auxin was weak and in WT plants appeared to be completely over-ridden by DELLA regulation. For a key GA deactivation (2-oxidase) gene, PsGA2ox1, the up-regulation induced by auxin deficiency was reduced to some extent by DELLA regulation. A second pea 2-oxidase gene, PsGA2ox2, was up-regulated by auxin, in a DELLA-independent manner. In Arabidopsis also, one 2-oxidase gene was down-regulated by auxin while another was up-regulated. Monitoring the metabolism pattern of GA₂₀ showed that in Arabidopsis, as in pea, auxin can promote the accumulation of bioactive GA.     

Analysis of tissue cadmium distribution in chronic cadmium-exposed mice using in-air micro-PIXE

This study undertook the analysis of tissue cadmium (Cd) distribution using in-air micro-particle-induced X-ray emission (PIXE) and the examination of the involvement of metal ions in parenteral Cd toxicity. A mouse was injected intraperitoneally with 3 mg/kg body weight of CdCl₂ thrice weekly. After 27 wk, the liver and kidney were excised and fixed in 10% formalin solution for 4 h and then embedded in paraffin. Thin paraffin sections were used to analyze trace elements with in-air micro-PIXE and to examine metallothionein protein and histological changes. Cd distribution was determined by micro-PIXE in the liver and renal cortex of the Cd-exposed mouse, and the net Cd count was higher in the liver than in the renal cortex. The net iron (Fe) count was higher in the liver of the Cd-exposed mouse compared to the control, and an opposite tendency was observed in the renal cortex. Wide cellular Cd distribution was demonstrated in the liver and renal cortex of the chronic Cd-exposed mouse compared to the control. Metallothionein staining was increased by chronic exposure to Cd both in the liver and kidney, and nephrotoxicity was more apparent than hepatotoxicity. The modification of tissue Fe and calcium distribution by an intraperitoneal injection of Cd might be involved in Cd-induced toxicity.     

Transcriptional Induction of Two Genes for CCaPs, Novel Cytosolic Proteins, in Arabidopsis thaliana in the Dark

Ca2+-signaling in downstream effectors is supported by many kinds of Ca2+-binding proteins, which function as a signal mediator and a Ca2+-buffering protein. We found in Arabidopsis thaliana a new type of Ca2+-binding protein, CCaP1, which consists of 152 amino acid residues, and binds (45)Ca2+ even in the presence of a high concentration of Mg2+. We found two other proteins with similar motifs, CCaP2 and CCaP3. These three proteins had no organelle localization signal and their green fluorescent protein (GFP) fusions were detected in the cytosol. Real-time PCR and histochemical analysis of promoter-beta-glucuronidase fusions revealed that CCaP1 was predominantly expressed in petioles while CCaP2 was expressed in roots. CCaP3 was hardly expressed. Expression of CCaP1 and CCaP2 was enhanced in darkness and became maximal after 24 h. Immunoblotting revealed petiole-specific accumulation of CCaP1. Expression of CCaP1 and CCaP2 was suppressed by a high concentration of Ca2+ and other metal ions. Deletion of sucrose from the medium markedly increased the mRNA levels of CCaP1 and CCaP2 within 2 h. Gibberellic acid enhanced the expression of CCaP1 and CCaP2 by 5- and 2.5-fold, respectively, after 6 h. CCaP1 and CCaP2 were suppressed in the petiole and the root, respectively, by light and the product of photosynthesis (sucrose) or both. These results suggest that CCaP1 functions as a mediator in response to continuous dark or gibberellic acid.     

Muscarinic potassium channels augment dynamic and static heart rate responses to vagal stimulation

Vagal control of heart rate (HR) is mediated by direct and indirect actions of ACh. Direct action of ACh activates the muscarinic K(+) (K(ACh)) channels, whereas indirect action inhibits adenylyl cyclase. The role of the K(ACh) channels in the overall picture of vagal HR control remains to be elucidated. We examined the role of the K(ACh) channels in the transfer characteristics of the HR response to vagal stimulation. In nine anesthetized sinoaortic-denerved and vagotomized rabbits, the vagal nerve was stimulated with a binary white-noise signal (0-10 Hz) for examination of the dynamic characteristic and in a step-wise manner (5, 10, 15, and 20 Hz/min) for examination of the static characteristic. The dynamic transfer function from vagal stimulation to HR approximated a first-order, low-pass filter with a lag time. Tertiapin, a selective K(ACh) channel blocker (30 nmol/kg iv), significantly decreased the dynamic gain from 5.0 +/- 1.2 to 2.0 +/- 0.6 (mean +/- SD) beats.min(-1).Hz(-1) (P < 0.01) and the corner frequency from 0.25 +/- 0.03 to 0.06 +/- 0.01 Hz (P < 0.01) without changing the lag time (0.37 +/- 0.04 vs. 0.39 +/- 0.05 s). Moreover, tertiapin significantly attenuated the vagal stimulation-induced HR decrease by 46 +/- 21, 58 +/- 18, 65 +/- 15, and 68 +/- 11% at stimulus frequencies of 5, 10, 15, and 20 Hz, respectively. We conclude that K(ACh) channels contribute to a rapid HR change and to a larger decrease in the steady-state HR in response to more potent tonic vagal stimulation.     

Simulation study on the disordered state of an Alzheimer's beta amyloid peptide Abeta(12 36) in water consisting of random-structural, beta-structural, and helical clusters

The monomeric Alzheimer's beta amyloid peptide, Abeta, is known to adopt a disordered state in water at room temperature, and a circular dichroism (CD) spectroscopy experiment has provided the secondary-structure contents for the disordered state: 70% random, 25% beta-structural, and 5% helical. We performed an enhanced conformational sampling (multicanonical molecular dynamics simulation) of a 25-residue segment (residues 12-36) of Abeta in explicit water and obtained the conformational ensemble over a wide temperature range. The secondary-structure contents calculated from the conformational ensemble at 300 degrees K reproduced the experimental secondary-structure contents. The constructed free-energy landscape at 300 degrees K was not plain but rugged with five clearly distinguishable clusters, and each cluster had its own characteristic tertiary structure: a helix-structural cluster, two beta-structural clusters, and two random-structural clusters. This indicates that the contribution from the five individual clusters determines the secondary-structure contents experimentally measured. The helical cluster had a similarity with a stable helical structure for monomeric Abeta in 2,2,2-trifluoroethanol (TFE)/water determined by an NMR experiment: The positions of helices in the helical cluster were the same as those in the NMR structure, and the residue-residue contact patterns were also similar with those of the NMR structure. The cluster-cluster separation in the conformational space indicates that free-energy barriers separate the clusters at 300 degrees K. The two beta-structural clusters were characterized by different strand-strand hydrogen-bond (H-bond) patterns, suggesting that the free-energy barrier between the two clusters is due to the H-bond rearrangements.     

Possible immune functions of congerin, a mucosal galectin, in the intestinal lumen of Japanese conger eel

Congerin, a mucosal galectin of the Japanese conger eel, provides chemical fortification through its agglutinating and opsonizing activity. Congerin is produced in the epidermis, and the epithelia of the oral cavity to the esophagus, but not in the stomach or intestine. We hypothesized that congerin secreted from the upper digestive tract can reach and function in the intestinal lumen. We found that congerin possessed marked resistance against digestion by gastric and enteric enzymes of conger eel. It was not degraded until 6h of incubation with stomach extract or intestinal digestion juice. Western blotting demonstrated that congerin essentially remained in the intestinal mucus. The mucus agglutinated rabbit erythrocytes, and the agglutination was hampered by anti-congerin antibody. Furthermore, congerin could bind to some enteric bacteria. These results support the above hypothesis.