Dithyreanitrile: an unusual insect antifeedant from Dithyrea wislizenii

Dithyreanitrile, a novel sulfur-containing indole alkaloid, was isolated from the seeds of Dithyrea wislizenii (Cruciferae). Dithyreanitrile inhibits feeding of fall armyworm (Spodoptera frugiperda) and European corn borer (Ostrinia nubilalis) larvae. Dithyreanitrile, the first natural product with two sulfur atoms and a nitrile attached to the same carbon, was characterized by X-ray diffraction, spectroscopy, and chemical synthesis.     

Mating patterns of virgin and inseminatedDrosophila melanogaster of different alcohol dehydrogenase (Adh) genotypes

Summary Female choice mating experiments showed virgin femaleD. melanogaster of the 3Adh genotypes chose heterozygous (Adh ^F /Adh ^S ) males most commonly. Inseminated females chose mates randomly, but the likelihood of a female remating was genotypically dependent.Acknowledgment. Financial support was provided by the Australian Research Grants Commitee.     

<Emphasis Type="Italic">LAPTM4A</Emphasis> interacts with <Emphasis Type="Italic">hOCT2</Emphasis> and regulates its endocytotic recruitment

Human organic cation transporter 2 (hOCT2) is involved in the transport of endogenous and exogenous organic cations mainly in cells of the kidney and the brain. Here, we focus on the regulation of hOCT2 by direct protein–protein interaction. Screening within a mating-based split-ubiquitin-yeast-two-hybrid system (mBSUS) revealed the lysosomal-associated protein transmembrane 4 alpha (LAPTM4A) as a potential interacting protein. Interaction of LAPTM4A and hOCT2 was confirmed by pulldown assays, FRET microscopy analysis and immunofluorescence microscopy. Functionally, overexpression of LAPTM4A significantly decreased ASP⁺ uptake in HEK293 cells stably transfected with hOCT2, suggesting a negative regulation of hOCT2-mediated transport. Furthermore, overexpression of LAPTM4A leads to a significantly decreased hOCT2 plasma membrane expression in surface biotinylation experiments. In addition, significant expression of LAPTM4A in human kidney was demonstrated by immunoblotting and immunofluorescence.     

Erythropoietic effects of PGE2 and 2 endoperoxide analogs

Summary The erythropoietic effects in exhypoxic polycythemic mice of two endoperoxide analogs were assessed and compared with PGE₂. The 9a, 11a epoxymethano analog (U-44069) was found to be a much more potent erythropoietic stimulus than the 11a,9a analog (U-46619) or PGE₂.This work was supported by a Senior Research Grant-in-Aid from the American Heart Association-Louisiana (DMG) and USPHS Grant No. AM 13211 (JWF).     

Kinetik der hepatischen Farbstoffaufnahme von Indocyaningrün. Einfluss von Bilirubin und Natriumglykocholat

Summary Kinetics of hepatic uptake of indocyanine green, a dye which is used for evaluation of liver function, were studied in the rat. The results indicate that the relationship between ICG-dose and initial hepatic dye uptake obeys Michaelis-Menten kinetics suggesting an interaction of the dye with a carrier or fixed site in the liver cell. Thus it was possible to calculate maximum ICG-uptake (v _max ) and the Michaelis constant (K _m ) of this transport system from several submaximal values.v _max was 7.65 (6-06-9.65)²² mg per 100 g liver/min and K _m 0.56 (0.31–0.81)²². Under the influence of substances which inhibit the elimination of dyes by the liver the parametersv _max and K _m showed changes which allowed characterization of the type of inhibition. While sodium glycocholate had no influence on maximum hepatic ICG-uptake and the Michaelis constant bilirubin caused a significant increase of K _m to 1.29 (0.68–1.90)²² without significantly changingv _max . These data suggest that bilirubin interferes with hepatic uptake of indocyanine green by competitive inhibition and that uptake of bile acids is dependent on a different mechanism.     

DNG1, a Dictyostelium homologue of tumor suppressor ING1 regulates differentiation of Dictyostelium cells

dng1 is a Dictyostelium homologue of the mammalian tumor suppressor ING gene. DNG1 protein localizes in the nucleus, and has a highly conserved PHD finger domain found in chromatin-remodeling proteins. Both dng1 disruption and overexpression impaired cell proliferation. In dng1-null cells, the progression of differentiation was delayed in a cell-density-dependent manner, and many tiny aggregates were formed. Exogenously applied cAMP pulses reversed the inhibitory effect caused by dng1 disruption on the aggregation during early development, but formation of tiny aggregates was not restored. dng1-overexpressing cells acquired the ability to undergo chemotaxis to cAMP earlier and exhibited enhanced differentiation. These phenotypes were found to be coupled with altered expressions of early genes such as cAMP receptor 1 (car1) and contact site A (csA). Furthermore, disordered histone modifications were demonstrated in dng1-null cells. These results suggest a regulatory role of dng1 in the transition of cells from growth to differentiation.Received 29 December 2004; received after revision 24 May 2005; accepted 26 May 2005     

A comparison of cycloartenol and lanosterol biosynthesis and metabolism by the Oomycetes

Summary Representative members in each of the four orders of Oomycetes (Phytophthora cactorum, Peronosporales;Lagenidium callinectes, L. giganteum, Lagenidiales;Saprolegnia ferax, Saprolegniales;Apodachylella completa, Leptomitales) have been examined for their ability to synthesize and polycyclize squalene-oxide (SO) to a tetracyclic product and to differentiate between cycloartenol and lanosterol metabolism to sterols.P. cactorum andL. giganteum failed to synthesize or metabolize SO, cycloartenol or lanosterol. While the other three fungi synthesized sterols via SO and lanosterol, a minor metabolism of added cycloartenol to the 4,4-desmethyl-14-methylcyclosteroid dehydropollinastanol was observed.Acknowledgment.Saprolegnia ferax-ATCC 3605 (1),Lagenidium callinectes ATCC 24973 (2),Apodachlyella completa (3) were obtained from Dr J. Aronson, Arizona State Univ. (3),Lagenidium gigateum was obtained from a drainage ditch in California (4), andPhytophthora cactorum was obtained from the U.C. Berkeley Fungal Collection (5). The fungi were cultured as previously described: Cultures 1,2,3: Berg, L. B., Ph. D. dissertation, Univ. of MD., College Park (1983); culture 4: Kerwin, J.L., and Washino, R.K., Exp. Mycol.7 (1983) 109; culture 5: Nes, W.D., and Stafford, A.E., Lipids19 (1984) 544. Preliminary observations involving the labeled substrates were presented by Le, P.H., Nes, W.D., and Parish, E.J. J. Am. Oil Chem. Soc.62 (1985) 655(A). Please address all correspondence to W.D. Nes, Plant Physiology and Chemistry Research Unit, ARS-US Dept of Agriculture, Berkeley, CA 94710, USA.     

Notable diversity in hemoglobin expression patterns among species of the deep-sea clam, <Emphasis Type="Italic">Calyptogena</Emphasis>

The deep-sea clams Calyptogena nautilei and C. tsubasa, which live in the cold-seep area at a depth of 3570 m in the Nankai Trough, Japan, have abundant hemoglobins (Hbs) in erythrocytes, similar to other Calyptogena species. We determined the cDNA-derived amino acid sequences of Hbs from two Calyptogena species. C. tsubasa was found to contain two dimeric Hbs, Hb I consisting of 145 amino acid residues and Hb II with 137 residues, similar to known Hbs from C. soyoae and C. kaikoi. Sequence identity was over 90% among the orthologous chains of Calyptogena Hbs. On the other hand, surprisingly, C. nautilei contained two monomeric Hbs, Hb III containing 141 residues and Hb IV with 134 residues. In addition, Hbs III and IV showed only 33–42% sequence identity with Hbs I and II from other Calyptogena species. The distal (E7) histidine, one of the functionally important residues of the heme protein, is replaced by glutamine in all Hb chains of Calyptogena species. A phylogenetic analysis indicated that C. nautilei Hb III is closer to Hb I from other Calyptogena species. We suppose that a Hb gene was duplicated at least three times in an immediate ancestor of Calyptogena and, presumably depending on physiological conditions different Hb sets are being expressed: dimeric Hbs I and II in C. soyoae, C. kaikoi and C. tsubasa, and monomeric Hbs III and IV in C. nautilei. Received 13 May 2003; received after revision 5 June 2003; accepted 12 June 2003     

Molecular characterization and expression of the antimicrobial peptide defensin from the housefly (Musca domestica)

A 430-bp cDNA encoding the insect antimicrobial peptide defensin was cloned from the housefly, and designated Musca domestica defensin (Mdde). The open reading frame of the cDNA encoded a 92-amino acid peptide with an N-terminal signal sequence followed by a propeptide that is processed by cleavage to a 40-amino acid mature peptide. Northern analysis and in situ hybridization identified the corresponding mRNA in the fat body of bacterially challenged houseflies and in the epidermis of the body wall of naive and challenged houseflies. The Gram-negative bacterium (Escherichia coli) is a strong inducer of the gene. By RT-PCR, Mdde mRNA was also detected in naive and challenged insects. These findings suggest that the defensin gene is constitutively expressed in the epidermis of the housefly body wall. The predicted mature form of Mdde was expressed as a recombinant peptide in E. coli and Pichia pastoris. The recombinant Mdde expressed in Pichia was active against Gram-positive and some Gram-negative bacteria. Received 20 June 2006; received after revision 3 October 2006; accepted 30 October 2006     

Periplasmic lysozyme inhibitor contributes to lysozyme resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The product of the Escherichia coli ORFan gene ykfE was recently shown to be a strong inhibitor of C-type lysozyme in vitro. The gene was correspondingly renamed ivy (inhibitor of vertebrate lysozyme), but its biological function in E. coli remains unknown. In this work, we investigated the role of Ivy in the resistance of E. coli to the bactericidal effect of lysozyme in the presence of outer-membrane-permeabilizing treatments. Both in the presence of lactoferrin (3.0 mg/ml) and under high hydrostatic pressure (250 MPa), the lysozyme resistance of E. coli MG1655 was decreased by knock-out of Ivy, and increased by overexpression of Ivy. However, knock-out of Ivy did not increase the lysozyme sensitivity of an E. coli MG1655 mutant previously described to be resistant to lysozyme under high pressure. These results indicate that Ivy is one of several factors that affect lysozyme resistance in E. coli, and suggest a possible function for Ivy as a host interaction factor in commensal and pathogenic E. coli.Received 12 February 2004; received after revision 11 March 2004; accepted 16 March 2004     

Biomedicine and diseases: the Klippel-Trenaunay syndrome, vascular anomalies and vascular morphogenesis

Vascular morphogenesis is a vital process for embryonic development, normal physiologic conditions (e.g. wound healing) and pathological processes (e.g. atherosclerosis, cancer). Genetic studies of vascular anomalies have led to identification of critical genes involved in vascular morphogenesis. A susceptibility gene, VG5Q (formally named AGGF1), was cloned for Klippel-Trenaunay syndrome (KTS). AGGF1 encodes a potent angiogenic factor, and KTS-associated mutations enhance angiogenic activity of AGGF1, defining ‘increased angiogenesis’ as one molecular mechanism for the pathogenesis of KTS. Similar studies have identified other genes involved in vascular anomalies as important genes for vascular morphogenesis, including TIE2, VEGFR-3, RASA1, KRIT1, MGC4607, PDCD10, glomulin, FOXC2, NEMO, SOX18, ENG, ACVRLK1, MADH4, NDP, TIMP3, Notch3, COL3A1 and PTEN. Future studies of vascular anomaly genes will provide insights into the molecular mechanisms for vascular morphogenesis, and may lead to the development of therapeutic strategies for treating these and other angiogenesis-related diseases, including coronary artery disease and cancer.Received 24 November 2004; received after revision 21 January 2005; accepted 2 March 2005     

Nitrate fertilizers as environmental pollutants: Positive correlation between nitrates (NaNO3 and KNO3) used per unit area and stomach cancer mortality rates

Summary Since a significant positive correlation between nitrate fertilizer exposure and stomach cancer mortality has been found, a study of nitrate fertilizer pollution in arable land in terms of amount of nitrates used per unit area (kg/ha) and stomach cancer death rates was carried out. The regression of death rates for stomach cancer, standardized for age, on the nitrates used per unit area, by province (N=25), exhibited a significant association between the 2 variables (p<0.004). When industrialized provinces (N=3) were not considered, a highly significant association was found (F=16.47; d.f.=1,20;p<0.0006). The correlation coefficient was+0.672.     

EGF-induced programmed cell death of human mammary carcinoma MDA-MB-468 cells is preceded by activation of AP-1

MDA-MB-468 is a human mammary adenocarcinoma cell line that overexpresses the epidermal growth factor (EGF) receptor and undergoes programmed cell death (apoptosis) in response to EGF treatment. Programmed cell death was shown to be greatly enhanced when cells were growth-arrested prior to EGF treatment. Apoptosis was characterized by an initial rounding up and detachment of the cells from their substrate starting about 12 h after EGF treatment, followed by chromatin condensation, nuclear fragmentation and oligonucleosomal fragmentation of the DNA at about 24 to 48 h. Cell death was dependent on de novo protein synthesis. We found a rapid induction of c-fos, c-jun and junB at the mRNA level after about 30 min of EGF treatment and a more delayed upregulation of fosB and fra-1. The junD gene was expressed in the absence of EGF, and it was moderately induced within 30 min of growth factor addition. The increase of the different fos and jun mRNAs were paralleled by an increase of activator protein-1 (AP-1) DNA binding activity. A characterization of the AP-1 complex revealed similar levels of several Fos and Jun proteins. Based on the kinetics of AP-1 accumulation and cell death, it seems likely that AP-1 contributes to the apoptotic cell death of EGF receptor-overexpressing MDA-MB-468 cells. Received 21 July 1997; received after revision 6 November 1997; accepted 6 November 1997     

Beta-carotene affects gene expression in lungs of male and female <Emphasis Type="Italic">Bcmo1</Emphasis><Superscript>−/−</Superscript> mice in opposite directions

Molecular mechanisms triggered by high dietary beta-carotene (BC) intake in lung are largely unknown. We performed microarray gene expression analysis on lung tissue of BC supplemented beta-carotene 15,15′-monooxygenase 1 knockout (Bcmo1 ^−/−) mice, which are—like humans—able to accumulate BC. Our main observation was that the genes were regulated in an opposite direction in male and female Bcmo1 ^−/− mice by BC. The steroid biosynthetic pathway was overrepresented in BC-supplemented male Bcmo1 ^−/− mice. Testosterone levels were higher after BC supplementation only in Bcmo1 ^−/− mice, which had, unlike wild-type (Bcmo1 ^+/+) mice, large variations. We hypothesize that BC possibly affects hormone synthesis or metabolism. Since sex hormones influence lung cancer risk, these data might contribute to an explanation for the previously found increased lung cancer risk after BC supplementation (ATBC and CARET studies). Moreover, effects of BC may depend on the presence of frequent human BCMO1 polymorphisms, since these effects were not found in wild-type mice.     

On the question of American eels,Anguilla rostrata versus European eels,Anguilla anguilla

Zusammenfassung Der amerikanische AalAnguilla rostrata (2n=38; 20M+18A) gehört einer anderen Spezies an als der europäischeAnguilla anguilla (2n=38; 32M+6A).

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This work was supported in part by a grant No. CA 05138 from the National Cancer Institute, U.S. Public Health Service.     

A potent attractant of zoospores ofAphanomyces cochlioides isolated from its host,Spinacia oleracea

A highly potent attractant of zoospores ofAphanomyces cochlioides, a causal fungus of the root rot disease of spinach (Spinacia oleracea), was isolated from spinach roots, and its structure was determined by spectroscopic evidence and chemical synthesis as cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone,1). A chromosorb particle prepared by soaking in solution of1 showed a potent attracting activity toward the zoospores using concentrations of1 above 10^–9 or 10^–10 M.     

Arachidonic acid release by ionomycin and phorbol ester is similar in C127 epithelial cells expressing wild-type or mutated (ΔF508) cystic fibrosis transmembrane conductance regulator

The Ca²⁺ ionophore ionomycin induced cytosolic [Ca²⁺]_i elevation as well as strong activation of Cl⁻ efflux in mouse mammary epithelial cell lines expressing wild-type or mutated (deletion of phenylalaline 508) cystic fibrosis transmembrane conductance regulator (CFTR) or vector. Ionomycin-induced Cl⁻ efflux was abolished by the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, whereas both activators and inhibitors of phospholipase A₂ had no effect, indicating the involvement of Ca²⁺-dependent Cl^- channels. Stimulation of arachidonic acid release by ionomycin and phorbol ester was not significantly different between wild-type or mutated cell lines, whereas vector-transfected cells exhibited a significant higher release, which was shown to be due to larger amount of immunoreactive cytosolic phospholipase A₂. These results indicate that phospholipase A₂ activity of C127 cells was not influenced by the presence of wild-type or mutated CFTR. Received 27 April 1999; received after revision 11 June 1999; accepted 23 July 1999     

Effect of benzimidazole on nicotinamide adenine dinucleotide phosphate phosphomonosterase activity in wheat leaves

Summary Nicotinamide adenine dinucleotide phosphate phosphomonoesterase was isolated and partially purified from wheat (Triticum aestivum L. var. Selkirk) leaves. The enzyme hadK _NADP value of 1.4×10^–4 M and a pH optimum of 5.9.In vitro activity of this enzyme was unaffected by precursors of NAD (nicotinamide and nicotinic acid) or cytokinis (kinetin and benzimidazole). However, when detached wheat leaves were treated with solutions of these compounds, the precursors lowered the specific activity while the cytokinins enhanced the activity. It is suggested that spatial separation and compartmentation of the enzyme and its substrate NADP account for the similar effect of benzimidazole on both.This work was supported by a grant No. A2698 from the National Research Council, Canada.