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Detection of seven virulence and toxin genes of Campylobacter jejuni isolates from Danish turkeys by PCR and cytolethal distending toxin production of the isolates

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JOURNAL OF FOOD PROTECTION 2004年第10期67卷 2171-2177页

作者： Bang, DD Borck, B Nielsen, EM Scheutz, F Pedersen, K Madsen, M Danish Vet Inst Dept Poultry Fish & Fur Anim DK-8200 Aarhus N Denmark Statens Serum Inst Dept Bacteriol Mycol & Parasitol DK-2300 Copenhagen S Denmark Statens Serum Inst Int Escherichia & Klebsiella Ctr DK-2300 Copenhagen Denmark

A total of 117 Campylobacter jejuni isolates from Danish turkeys were tested for the presence of seven virulence and toxin genes by PCR. One hundred seventeen (100%) isolates were positive for flaA, cadF, and ceuE gene primers. One hundred three (88%) isolates were positive for cdt gene cluster PCR detection (cdt gene cluster-PCR), whereas 101 (86.3%), 102 (87.2%), and 110 (94%) isolates were positive for cdtA-, cdtB-, and cdtC-PCR, respectively. Only 39 (33.3%) isolates were positive for virB11. Of 1.17 isolates, 114 (97.4%) produced cytolethal distending toxin (CDT) in Vero cell assays, 105 (89.7%) in Colon 205 assays, and 109 (93.2%) in chicken embryo cell assays. The CDT titers were determined in Vero cell assays. Of 117 isolates, 50 (42.7%) produced a CDT titer of 1:100, 29 (24.8%) of 1:50, and 27 (23%) of 1:5 to 1:10;8 (6.8%) produced a CDT titer at undiluted supernatants and 3 (2.6%) produced no toxin. Twenty-nine C. jejuni isolates that were PCR negative for one or more individual cdt toxin genes also produced low or no CDT toxin. The high prevalence of the seven virulence and toxin genes demonstrates that these putative pathogenic determinants are widespread among Campylobacter isolates from turkeys and calls for further investigation for the elimination of Campylobacter infection in industrial turkey production and in industrial food chains.

关键词： Animals Bacterial Toxins genetics Campylobacter Infections microbiologyveterinary Campylobacter jejuni genetics pathogenicity cercopithecus aethiops Chick Embryo Chickens Denmark epidemiology Genes Bacterial Human Polymerase Chain Reaction Poultry Diseasesmicrobiology Support Non-U.S. Gov't Turkeys microbiology Vero Cells Virulence genetics

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An ear-core interaction regulates the recruitment of the AP-3 complex to membranes

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DEVELOPMENTAL CELL 2004年第4期7卷 619-625页

作者： Lefrançois, S Janvier, K Boehm, M Ooi, CE Bonifacino, JS NICHHD Cell Biol & Metab Branch NIH Bethesda MD 20892 USA

AP-3 is a heterotetrameric adaptor involved in the biogenesis of lysosome-related organelles. The function of AP-3 as an adaptor relies on its ability to bind to membranes in an Art-dependent fashion and to recognize sorting signals in the cytosolic tails of the transmembrane cargo. Here, we report an interdomain interaction involving the ear domain of the delta subunit and the sigma3 subunit of AP-3. This interaction interferes with the binding of AP-3 to Art but not to dileucine-based sorting signals. As a consequence, the delta-ear inhibits the recruitment of AP-3 to membranes both in vitro and in vivo and impairs the sorting of lysosomal membrane proteins. These observations suggest a new regulatory mechanism for the recruitment of AP-3 to membranes involving delta-ear-sigma3 interactions.

关键词： Amino Acid Sequence Amino Acid Substitution Animals COS Cells Cell Extracts cercopithecus aethiops Clone Cells Endosomes Flow Cytometry Glutathione Transferase Hela Cells Humans Membranes Models Biological Protein Conformation Protein Structure Tertiary Protein Subunits Protein Transport Recombinant Fusion Proteins Research Support Non-U.S. Gov't Transcription Factors inhibitors Two-Hybrid System Techniques

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Huntingtin-interacting protein HIP14 is a palmitoyl transferase involved in palmitoylation and trafficking of multiple neuronal proteins

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NEURON 2004年第6期44卷 977-986页

作者： Huang, K Yanai, A Kang, R Arstikaitis, P Singaraja, RR Metzler, M Mullard, A Haigh, B Gauthier-Campbell, C Gutekunst, CA Hayden, MR El-Husseini, A Womens & Childrens Hosp Dept Med Genet Ctr Mol Med & Therapeut Vancouver BC Canada Univ British Columbia Dept Psychiat Brain Res Ctr Vancouver BC V5Z 1M9 Canada Emory Univ Dept Neurol Atlanta GA 30329 USA

In neurons, posttranslational modification by palmitate regulates the trafficking and function of signaling molecules, neurotransmitter receptors, and associated synaptic scaffolding proteins. However, the enzymatic machinery involved in protein palmitoylation has remained elusive. Here, using biochemical assays, we show that huntingtin (htt) interacting protein, HIP14, is a neuronal palmitoyl transferase (PAT). HIP14 shows remarkable substrate specificity for neuronal proteins, including SNAP-25, PSD-95, GAD65, synaptotagmin I, and htt. Conversely, HIP14 is catalytically invariant toward paralemmin and synaptotagmin VII. Exogenous HIP14 enhances palmitoylation-dependent vesicular trafficking of several acylated proteins in both heterologous cells and neurons. Moreover, interference with endogenous expression of HIP14 reduces clustering of PSD-95 and GAD65 in neurons. These findings define HIP14 as a mammalian palmitoyl transferase involved in the palmitoylation and trafficking of multiple neuronal proteins.

关键词： Acyltransferases Adaptor Proteins, Signal Transducing Amino Acid Sequence Animals Carnitine O-Palmitoyltransferase/genetics Carnitine O-Palmitoyltransferase/metabolism Carnitine O-Palmitoyltransferase/physiology Carrier Proteins/genetics Carrier Proteins/metabolism Carrier Proteins/physiology Cells, Cultured cercopithecus aethiops COS Cells Humans Molecular Sequence Data Nerve Tissue Proteins/genetics Nerve Tissue Proteins/metabolism Nerve Tissue Proteins/physiology Palmitic Acid/metabolism Protein Transport/physiology Substrate Specificity

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Antiviral and antiplasmodial spirodihydrobenzofuran terpenes from the fungus Stachybotrys nephrospora

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PLANTA MEDICA 2004年第11期70卷 1085-1087页

作者： Sawadjoon, S Kittakoop, P Isaka, M Kirtikara, K Madla, S Thebtaranonth, Y NSTDA Natl Ctr Genet Engn & Biotechnol BIOTEC Klongluang 12120 Pathumthani Thailand Mahidol Univ Fac Sci Dept Chem Bangkok 10400 Thailand

Two known spirodihydrobenzofuran terpenes (1 and 2) were isolated from a mycelium extract of the fungus Stachybotrys nephrospora BCC 3900. Compound 1 (Mer-NF5003F or stachybotrydial) exhibited potent antiviral activity (the IC50 value of 4.32 mug/mL) comparable to the standard drug, acyclovir, while compound 2 was inactive against the HSV-1 virus. Both 1 and 2 possessed antiplasmodial activity (IC50 values of 0.85 and 0.15 mug/mL for 1 and 2, respectively), and were not toxic towards the Vero cell line. A regiospecific conversion of the dialdehyde 1 to the lactone 2 proceeded simply under acidic conditions.

关键词： Phytotherapy Stachybotrys Animals Antimalarials Antiviral Agents cercopithecus aethiops Herpes Simplex Herpesvirus 1 Human Humans Malaria Falciparum Microbial Sensitivity Tests Parasitic Sensitivity Tests Plant Extracts Plasmodium falciparum Research Support Non-U.S. Gov't Vero Cells

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Toxicokinetics of ochratoxin A in vervet monkeys (cercopithecus aethiops)

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ARCHIVES OF TOXICOLOGY 2001年第5期75卷 262-269页

作者： Stander, MA Nieuwoudt, TW Steyn, PS Shephard, GS Creppy, EE Sewram, V Univ Stellenbosch Dept Chem ZA-7602 Matieland South Africa Potchefstroom Univ Christian Higher Educ Sch Chem & Biochem ZA-2520 Potchefstroom South Africa MRC PROMEC ZA-7505 Tygerberg South Africa Univ Bordeaux 2 Lab Toxicol & Hyg Appliquee F-33076 Bordeaux France

The toxicokinetics of ochratoxin A were investigated in vervet monkeys (cercopithecus aethiops). Three female monkeys were treated intravenously with ochratoxin A at doses, respectively, of 0.8, 1.5 and 2 mg/kg body weight (BW). Blood and urine samples were collected over a period of 21 days. Plasma and urine extracts were analysed by high-performance liquid chromatography (HPLC) with either fluorescence or negative ion electrospray ionization mass spectrometric detection. The clearance of ochratoxin A from plasma followed a two-compartment model. The elimination half-life of ochratoxin A in the monkeys was determined to be 19-21 days and the average total body clearance was 0.22 +/-0.07 ml/h per ka, and the average apparent distribution volume of the central compartment was 59 +/-9 ml/kg and the peripheral compartment was 59 +/- 20 ml/kg. No evidence was found for any metabolic conversion of ochratoxin A.

关键词： ochratoxin A toxicokinetics cercopithecus aethiops intravenous negative ion electrospray ionization mass spectrometry

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Penetratin and related cell-penetrating cationic peptides can translocate across lipid bilayers in the presence of a transbilayer potential

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BIOCHEMISTRY 2003年第47期42卷 13787-13799页

作者： Terrone, D Sang, SLW Roudaia, L Silvius, JR McGill Univ Dept Biochem Montreal PQ H3G 1Y6 Canada

Fluorescent-labeled derivatives of the Antennapedia-derived cell-penetating peptide penetratin, and of the simpler but similarly charged peptides R(6)GC-NH2 and K(6)GC-NH2, are shown to be able to translocate into large unilamellar lipid vesicles in the presence of a transbilayer potential (inside negative). Vesicles with diverse lipid compositions, and combining physiological proportions of neutral and anionic lipids, are able to support substantial potential-dependent uptake of all three cationic peptides. The efficiency of peptide uptake under these conditions is strongly modulated by the vesicle lipid composition, in a manner that suggests that more than one mechanism of peptide uptake may operate in different systems. Remarkably, peptide uptake is accompanied by only minor perturbations of the overall barrier function of the lipid bilayer, as assessed by assays of vesicle leakiness under the same conditions. Fluorescence microscopy of living CV-1 and HeLa cells incubated with the labeled peptides shows that the peptides accumulate in peripheral vesicular structures at early times of incubation, consistent with an initial endosomal localization as recently reported, but gradually accumulate in the cytoplasm and nucleus during more extended incubations (several hours). Our findings indicate that these relatively hydrophilic, polybasic cell-penetrating peptides can translocate through lipid bilayers by a potential- and composition-dependent pathway that causes only minimal perturbation to the overall integrity and barrier function of the bilayer.

关键词： Amino Acid Sequence Animals Carrier Proteins Cell Line Cell Membrane Permeability cercopithecus aethiops Drosophila Proteins Hela Cells Homeodomain Proteins Human Lipid Bilayers Membrane Potentials Molecular Sequence Data Peptide Fragments Phosphatidylcholines Phosphatidylethanolamines Phosphatidylglycerols Protein Transport Support Non-U.S. Gov't Time Factors

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Inhibiting severe acute respiratory syndrome-associated coronavirus by small interfering RNA

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Chinese Medical Journal 2003年第8期116卷 1262-1264页

作者：张仁礼郭中敏陆家海孟锦绣周灿权詹希美黄冰余新炳黄民潘兴华凌文华陈系古万卓越郑焕英鄢心革王一飞冉延超刘新健马俊鑫王承宇张必良 Sun Yat-Sen University Center for Disease Control and Prevention of Guangdong Province Jinan University Maxim Biotech Inc. The Quartermaster University of PLA Medical School of University of Massachusetts

OBJECTIVE: To evaluate the effectiveness of small interfering RNA (siRNA) on inhibiting severe acute respiratory syndrome (SARS)-associated coronavirus replication, and to lay bases for the future clinical application of siRNA for the treatment of viral infectious diseases. METHODS: Vero-E6 cells was transfected with siRNA before SARS virus infection, and the effectiveness of siRNA interference was evaluated by observing the cytopathic effect (CPE) on Vero-E6 cells. RESULTS: Five pairs of siRNA showed ability to reduce CPE dose dependently, and two of them had the best effect. CONCLUSION: siRNA may be effective in inhibiting SARS-associated coronavirus replication.

关键词： Animals cercopithecus aethiops RNA, Small Interfering SARS Virus Transfection Vero Cells Virus Replication

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Melanopsin forms a functional short-wavelength photopigment

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BIOCHEMISTRY 2003年第44期42卷 12734-12738页

作者： Newman, LA Walker, MT Brown, RL Cronin, TW Robinson, PR Univ Maryland Baltimore Cty Dept Biol Sci Baltimore MD 21250 USA Oregon Hlth & Sci Univ Inst Neurol Sci Beaverton OR 97006 USA

Recently, melanopsin has emerged as the leading candidate for the elusive photopigment of the mammalian circadian system. This novel opsin-like protein is expressed in retinal ganglion cells that form the retinohypothalamic tract, a neuronal connection between the retina and the suprachiasmatic nucleus. These hypothalamic structures contain the circadian pacemaker, which generates daily rhythms in physiology and behavior. In mammals, proper synchronization of these rhythms to the environmental light-dark cycle requires retinal input. Surprisingly, rod and cone photoreceptors are not required. Instead, the melanopsin-containing ganglion cells are intrinsically sensitive to light, perhaps responding via a melanopsin-based signaling pathway. To test this hypothesis, we have characterized melanopsin following heterologous expression in COS cells. We found that melanopsin absorbed maximally at 424 nm after reconstitution with 11-cis-retinal. Furthermore, melanopsin activated the photoreceptor G-protein, transducin, in a light-dependent manner. In agreement with the measured absorbance spectrum, melanopsin was most efficiently excited by blue light (420-440 nm). In contrast, published action spectra suggest that the photopigment underlying the intrinsic light sensitivity of SCN-projecting RGCs has an absorption maximum near 484 nm. In summary, our experiments constitute the first direct demonstration that melanopsin forms a photopigment capable of activating a G-protein, but its spectral properties are not consistent with the action spectrum for circadian entrainment.

关键词： Amino Acid Sequence Animals COS Cells Cattle cercopithecus aethiops Light Mice Molecular Sequence Data Mutagenesis Site-Directed Opsin Photoperiod Retinal Ganglion Cells Signal Transduction Spectrophotometry Support U.S. Gov't Non-P.H.S. Support U.S. Gov't P.H.S. Transducin Transfection cis-trans-Isomerases

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Molecularly engineered live-attenuated chimeric West Nile/dengue virus vaccines protect rhesus monkeys from West Nile virus

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VIROLOGY 2003年第1期314卷 190-195页

作者： Pletnev, AG St Claire, M Elkins, R Speicher, J Murphy, BR Chanock, RM NIAID Infect Dis Lab NIH Bethesda MD 20892 USA Bioqual Inc Rockville MD 20852 USA

Two molecularly engineered, live-attenuated West Nile virus (WN) vaccine candidates were highly attenuated and protective in rhesus monkeys. The vaccine candidates are chimeric viruses (designated WN/DEN4) bearing the membrane precursor and envelope protein genes of WN on a backbone of dengue 4 virus (DEN4) with or without a deletion of 30 nucleotides (Delta30) in the 3' noncoding region of DEN4. Viremia in WN/DEN4- infected monkeys was reduced 100-fold compared to that in WN- or DEN4-infected monkeys. WN/DEN4-3'Delta30 did not cause detectable viremia, indicating that it is even more attenuated for monkeys. These findings indicate that chimerization itself and the presence of the Delta30 mutation independently contribute to the attenuation phenotype for nonhuman primates. Despite their high level of attenuation in monkeys, the chimeras induced a moderate-to-high titer of neutralizing antibodies and prevented viremia in monkeys challenged with WN. The more attenuated vaccine candidate, WN/DEN4-3'Delta30, will be evaluated first in our initial clinical studies. (C) 2003 Elsevier Inc. All rights reserved.

关键词： Animals Antibodies, Viral/blood cercopithecus aethiops Dengue Virus/genetics Dengue Virus/immunology Dengue Virus/metabolism Disease Models, Animal Genetic Engineering/methods Humans Macaca mulatta Neutralization Tests Neutralization Tests Recombinant Fusion Proteins/genetics Recombinant Fusion Proteins/immunology Vaccines, Attenuated/administration & dosage Vaccines, Attenuated/genetics Vaccines, Attenuated/immunology Vaccines, Synthetic/administration & dosage Vaccines, Synthetic/genetics Vaccines, Synthetic/immunology Vero Cells Viral Envelope Proteins/genetics Viral Envelope Proteins/immunology Viral Envelope Proteins/metabolism Viral Vaccines/administration & dosage Viral Vaccines/genetics Viral Vaccines/immunology West Nile Fever/prevention & control West Nile virus/genetics West Nile virus/immunology West Nile virus/metabolism

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Structure-activity relationships of (S,Z)-2-aminopurine methylenecyclopropane analogues of nucleosides.: Variation of purine-6 substituents and activity against herpesviruses and hepatitis B virus

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JOURNAL OF MEDICINAL CHEMISTRY 2003年第8期46卷 1531-1537页

作者： Chen, XC Kern, ER Drach, JC Gullen, E Cheng, YC Zemlicka, J Wayne State Univ Sch Med Dept Chem Dev Therapeut ProgramBarbara Ann Karmanos Canc In Detroit MI 48201 USA Univ Alabama Dept Pediat Birmingham AL 35294 USA Univ Michigan Sch Dent Dept Biol & Mat Sci Ann Arbor MI 48019 USA Yale Univ Sch Med Dept Pharmacol New Haven CT 06510 USA

A series of 13 new (S,Z)-2-aminopurine methylenecyclopropane analogues was synthesized, and their antiviral activity was investigated. The nucleophilic displacement of chlorine of 2-amino-6-chloropurine derivative 5 with allyl-, propargyl-, cyclopropylmethyl-, isopropyl-, benzyl-, cyclohexyl-, and 2-hydroxyethylamine gave N-6-alkyl compounds 2a, 2b, 2c, 2d, 2e, 2f, and 2g. A similar reaction of 5 with allyl, cyclopropylmethyl, propyl, or pentyl alcohol catalyzed by K2CO3 afforded O-6-alkyl analogues 3a, 3c, 3h and 3i. Propane- and pentanethiol furnished S-6-alkyl compounds 4h and 4i. The N-6-alkyl derivatives 2a, 2b, O-6 analogues 3a, 3c, 3h, 3i, and S-6 compounds 4h, 4i which were highly effective in all CMV assays and exhibited the lowest cytotoxicity in proliferating HFF cells appear to be good candidates for in vivo assays. Activity of new analogues against HSV-1 or HSV-2 was restricted to BSC-1 and Vero cultures. Compounds 2c, 2b, 3a and 3h were effective against EBV in one of two assays (Daudi or H-1). Analogues 3a and 4i were the most active anti-VZV agents whereas compounds 3h, 3i, and 4h inhibited the replication of HBV in a micromolar concentration range.

关键词： Aminopyrine Animals Antiviral Agents Cell Line cercopithecus aethiops Cyclopropanes Enzyme-Linked Immunosorbent Assay Hepatitis B Virus Herpesviridae Human Mice Plaque Assay Purine Nucleosides Stereoisomerism Structure-Activity Relationship Support U.S. Gov't P.H.S.

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