bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on exudate-depleted sclerotial development in Sclerotium rolfsii and the effect of oxalic acid, sclerotial exudate, and culture filtrate on phenolic acid induction in chickpea (Cicer arietinum)
Authors: Singh U P , Sarma B K , Singh D P , Bahadur Amar
Published: October 2002

Exudate depletion from developing sclerotia of Sclerotium rolfsii Sacc. in culture caused reduced size and weight of sclerotia. Germination of exudate-depleted sclerotia was delayed on Cyperus rotundus rhizome meal agar medium when compared with that of control sclerotia. The exudate-depleted sclerotia caused infection in chickpea (Cicer arietinum) plants in a glasshouse. Different temperatures and incubation periods had no effect on the germination ability of the exudate-depleted sclerotia. Oxalic acid, sclerotial exudate, and culture filtrate of S. rolfsii induced the synthesis of phenolic acids, including gallic, ferulic, chlorogenic, and cinnamic acids, as well as salicylic acid, in treated chickpea leaves. Gallic acid content was increased in treated leaves compared with the untreated controls. Maximum induction of gallic acid was seen in both leaves treated with oxalic acid followed by exudate and leaves treated with culture filtrate. Cinnamic and salicylic acids were not induced in exudate-treated leaves. Ethyl acetate fractionation indicated that the sclerotial exudates consisted of gallic, oxalic, ferulic, chlorogenic, and cinnamic acids, whereas the culture filtrate consisted of gallic, oxalic, and cinnamic acids along with many other unidentified compounds.Key words: oxalic acid, phenolic acid, salicylic acid, sclerotial exudate, culture filtrate, Sclerotium rolfsii, sclerotial germination.
Cryptococcus tephrensis, sp.nov., and Cryptococcus heimaeyensis, sp.nov.; new anamorphic basidiomycetous yeast species from Iceland
Authors: Vishniac Helen S
Published: October 2002

Two new species from Iceland are described on the basis of physiological profiles and sequence data from the D2 region of LSU rDNA: Cryptococcus tephrensis (type ICE99-1ToM Y5, ATCC MYA-1765, CBS 8935, GenBank AF317208) and Cryptococcus heimaeyensis (type ICE99-1ToM Y8, ATCC MYA-1759, CBS 8933, GenBank AF370717). The two new species are identifiable from sequence data and can be distinguished from their closest relative, Cryptococcus victoriae, by their higher maximum temperatures for growth, failure to utilize nitrate as sole nitrogen source, and utilization of cadaverine and ethylamine as sole nitrogen sources. Cryptococcus tephrensis is distinguishable from C. heimaeyensis by failure to grow on saccharate as sole source of carbon and energy.Key words: Iceland, Cryptococcus, soil yeasts.
Effect offurmutation on acid-tolerance response and in vivo virulence of avian septicemicEscherichia coli
Authors: Zhu Chengru , Ngeleka Musangu , Potter Andrew A , Allan Brenda J
Published: October 2002

The Fur (ferric uptake regulator) protein is a master regulator of iron metabolism in gram-negative bacteria. In the present study, the effect of a partial deletion of the fur gene on the acid-tolerance response and in vivo virulence of avian Escherichia coli was examined. The fur mutant was unable to trigger the acid-tolerance response as observed in the wild-type parent strain. However, the mutant was as virulent as the wild-type parent strain when tested in 1-day-old chickens by subcutaneous inoculation. These data indicate that the fur gene is involved in the acid-tolerance response but not involved in the virulence of E. coli, as detected by the ability to cause septicemia in our experimental infection.Key words: E. coli, fur, acid-tolerance response.
Occurrence of several genes encoding putative reductive dehalogenases inDesulfitobacterium hafniense/frappieriandDehalococcoides ethenogenes
Authors: Villemur Richard , Saucier Maude , Gauthier Annie , Beaudet Réjean
Published: October 2002

Desulfitobacterium frappieri PCP-1 has the capacity to dehalogenate several halogenated aromatic compounds by reductive dehalogenation, however, the genes encoding the enzymes involved in such processes have not yet been identified. Using a degenerate oligonucleotide corresponding to a conserved sequence of CprA/PceA reductive dehalogenases, a cprA-like gene fragment was amplified by PCR from this bacterial strain. A Delfitobacterium frappieri PCP-1 cosmid library was screened with the PCR product, allowing the cloning and sequencing of a 1.9-kb fragment. This fragment contains a nucleic acid sequence identical to one genomic contig of Desulfitobacterium hafniense, a bacterium closely related to Delfitobacterium frappieri that is also involved in reductive dehalogenation. Other genes related to the Desulfitobacterium dehalogenans cpr locus were identified in this contig. Interestingly, the gene arrangement shows the presence of two copies of cprA-, cprB-, cprC-, cprD-, cprK-, and cprT-related genes, suggesting that gene duplication occurred within this chromosomic region. The screening of Delfitobacterium hafniense genomic contigs with a CprA-deduced amino acid sequence revealed two other cprA-like genes. Microbial genomes available in gene databases were also analyzed for sequences related to CprA/PceA. Two open reading frames encoding other putative reductive dehalogenases in Delfitobacterium hafniense contigs were detected, along with 17 in the Dehalococcoides ethenogenes genome, a bacterium involved in the reductive dehalogenation of tetrachloroethene to ethene. The fact that several gene encoding putative reductive dehalogenases exist in Delfitobacterium hafniense, probably in other members of the genus Desulfitobacterium, and in Dehalococcoides ethenogenes suggests that these bacteria use distinct but related enzymes to achieve the dehalogenation of several chlorinated compounds.Key words: Desulfitobacterium, reductive dehalogenases, halorespiration, chlorinated compounds, gene family.
Physiological and genetic characterization of fluorescentPseudomonasassociated withCantharellus cibarius
Authors: Rangel-Castro J Ignacio , Levenfors Jolanta J , Danell Eric
Published: October 2002

Fluorescent Pseudomonas spp. isolated from fruiting bodies (FB) of Cantharellus cibarius were characterized physiologically and genetically and were compared with fluorescent Pseudomonas from forest soil and with sequences from the GenBank database. Pseudomonas spp. from FB differed physiologically from isolates from soil lacking FB and had some similarities with the strains obtained from soil underneath the FB. Analyses of the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) patterns and partial sequencing analysis of the 16S-rDNA region indicated that the bacteria isolated from these environments were different. However, there was no specific Pseudomonas genotype restricted to the FB environment. Utilization of the reported fungal exudates trehalose and mannitol may explain how millions of bacteria survive in the C. cibarius FB without deteriorating the fungal mycelium. The importance of the metabolic characterization of bacteria and the possible mechanisms involved in the association with C. cibarius are discussed. Our study showed that standard processes for bacterial identification, e.g., Biolog®and 16S-rDNA are insufficient until databases for different ecosystems are created.Key words: Cantharellus cibarius, fluorescent Pseudomonas, carbon utilization, PCRRFLP, 16S-rDNA sequencing.
Adhesion of Bacillus spores and Escherichia coli cells to inert surfaces: role of surface hydrophobicity
Authors: Faille Christine , Jullien Celine , Fontaine Francoise , Bellon-Fontaine Marie-Noelle , Slomianny Christian , Benezech Thierry
Published: October 2002

The ability of bacterial spores and vegetative cells to adhere to inert surfaces was investigated by means of the number of adherent spores (Bacillus cereus and Bacillus subtilis spores) and Escherichia coli cells and their resistance to cleaning or rinsing procedures (adhesion strength). Six materials (glass, stainless steel, polyethylene high density (PEHD), polyamide-6, polyvinyl chloride, and Teflon®) were tested. Slight differences in the number of adherent spores (less than 1 log unit) were observed between materials, but a higher number of adherent E. coli cells was found on the hydrophobic materials PEHD and Teflon®. Conversely, the resistance of both B. cereus and B. subtilis spores to a cleaning procedure was significantly affected by the material. Hydrophobic materials were harder to clean. The topography parameter derived from the Abbott-Firestone curve, RVK, and, to a lesser extent, the widely used roughness parameters RA (average roughness) and RZ (maximal roughness), were related to the number of adherent cells. Lastly, the soiling level as well as the adhesion strength were shown to depend largely on the microorganism. The number of adhering B. cereus hydrophobic spores and their resistance to a cleaning procedure were found to be 10 times greater than those of the B. subtilis hydrophilic spores. Escherichia coli was loosely bound to all the materials tested, even after 24 h biofilm formation.Key words: adhesion, cleaning, bacteria, spores, roughness, surface energies.
NoteIdentification of atypical strains of Malassezia spp. from cattle and dog
Authors: Duarte Eduardo Robson , Lachance Marc-André , Hamdan Júnia Soares
Published: October 2002

Yeast species in the genus Malassezia are lipophilic with the exception of Malassezia pachydermatis. During a study of the occurrence of Malassezia species in the external ear of 964 cattle and 6 dogs in Minas Gerais, Brazil, six lipid-dependent isolates could not be identified to known species. Four isolates came from healthy cows, one from a cow with otitis, and one from a healthy dog. When tested with Tweens and Cremophor EL as single sources of lipids, the strains grew on all sources except Cremophor EL. None of the six strains hydrolyzed esculin, and all produced catalase. Pigment production from tryptophan was variable. Partial large subunit rRNA sequences were obtained for two isolates that remained viable in culture. The strain from the cow with otitis was identified as a lipid-dependent variant of M. pachydermatis, and the strain from the dog was an atypical variant of Malassezia furfur.Key words: atypical Malassezia strains, biochemical and physiological characterization, ribosomal DNA.
Fermentation of chicory fructo-oligosaccharides in mixtures of different degrees of polymerization by three strains of bifidobacteria
Authors: Perrin S , Fougnies C , Grill J P , Jacobs H , Schneider F
Published: October 2002

We estimated and compared the action of three selected strains of bifidobacteria in a semi-synthetic medium for different degrees of polymerization of fructo-oligosaccharides contained in three commercial products derived from chicory inulin: Fibrulose®F97 (shorter chains), Fibruline®Instant (native chains), Fibruline®LC (longer chains). Biomass and production of lactate and acetate were greater when the substrate contained mostly shorter chain fructo-oligosaccharides. Shorter chains were first to be consumed, and one strain could use longer chains. As the degree of polymerization increased, residual fructo-oligosaccharides increased after growth of the strains, and the rate of consumption of fructo-oligosaccharides decreased.Key words: Bifidobacterium, fructo-oligosaccharides, inulin, oligofructose, fermentation.
Predicting survival of a genetically engineered microorganism, Pseudomonas chlororaphis 3732RN-L11, in soil and wheat rhizosphere across Canada with linear multiple regression models
Authors: Edge Thomas A , Wyndham R Campbell
Published: October 2002

Pseudomonas chlororaphis 3732RN-L11 survival rates in soil and wheat rhizosphere were measured using intact soil core microcosms representing 23 sites across Canada. Linear multiple regression (LMR) models were developed to predict the survival rate of this genetically engineered microorganism (GEM) as a function of soil parameters measured at the time of microcosm inoculation. LMR models were tested by comparing their predicted survival rates with observed survival rates from environmental introductions of the GEM by Gagliardi et al. (2001) at five field sites across Canada over two years. No soil parameter (e.g., % clay) was highly correlated with GEM survival rates in soil or wheat rhizosphere. Total fungal colony-forming units (CFUs), % soil titanium (positive correlations), and % soil magnesium (negative correlation) were found to be the best LMR predictors of GEM survival rates in soil over two years. Total soil bacterial CFUs, nitrate, % soil potassium (positive correlations), and exchangeable magnesium (negative correlation) were found to be the best LMR predictors of GEM survival rate in wheat rhizosphere over two years. While LMR models were statistically significant, they were unable to reliably predict the survival rate of the GEM in field trial introductions. The results indicate that there can be considerable uncertainty associated with predicting GEM survival for multi-site environmental introductions.Key words: Pseudomonas chlororaphis 3732RN-L11, survival, soil, Canada.
Agrobacterium flagellar switch gene fliG is liquid inducible and important for virulence
Authors: Li Luoping , Jia Yong H , Pan Shen Q
Published: October 2002

Agrobacterium tumefaciens C58 was mutagenized with a mini-Tn5 transposon containing a promoterless gene encoding the green fluorescent protein (GFP). A mutant, CGS74, exhibited a higher GFP expression level in liquid media than on solid media. The ability of the mutant to cause tumors on plants was attenuated. Sequence analysis showed that the transposon was inserted at the fliG gene, which encodes a flagellar motor switch protein required for flagellar movement. Studies of the fliGgfp fusion gene indicated that the promoter activity of the fliG gene was higher in liquid than in solid media. Electron microscopy studies demonstrated that the mutant was nonflagellate. This suggests that the A. tumefaciens motility is important for virulence and that bacterial flagellar synthesis occurs at a higher level in a liquid environment than in a solid environment, perhaps resulting in a higher motility.Key words: Agrobacterium, flagella, fliG, virulence, liquid.
Interspecies communication betweenBurkholderia cepaciaandPseudomonas aeruginosa
Authors: Lewenza Shawn , Visser Michelle B , Sokol Pamela A
Published: October 2002

Burkholderia cepacia and Pseudomonas aeruginosa are opportunistic pathogens that commonly cause pulmonary infections in cystic fibrosis patients and occasionally co-infect patients' lungs. Both organisms possess quorum-sensing systems dependent on N-acyl homoserine lactone (N-acyl-HSL). Cross-feeding assays demonstrated that P. aeruginosa and B. cepacia were able to utilize heterologous N-acyl-HSL signaling molecules. The ability of quorum-sensing genes from one species to complement the respective quorum-sensing mutations in the heterologous species was also examined. These studies suggest that B. cepacia CepR can use N-acyl-HSLs synthesized by RhlI and LasI and that P. aeruginosa LasR and RhlR can use N-acyl-HSLs synthesized by CepI. It is possible that a mixed bacterial population of B. cepacia and P. aeruginosa can coordinately regulate some of their virulence factors and influence the progression of lung disease due to infection with these organisms.Key words: quorum sensing, Burkholderia cepacia, Pseudomonas aeruginosa, cystic fibrosis.
Monitoring ofBacillus thermodenitrificansOHT-1 in compost by whole cell hybridization
Authors: Hatsu Masahiro , Ohta Junji , Takamizawa Kazuhiro
Published: October 2002

Thermophilic aerobic composting is a widely practiced method for the disposal of exhaust materials. We isolated a thermophilic bacteria strain from a compost sample under aerobic conditions at 60°C. On the basis of its 16S rRNA sequence and physiological characteristics, this strain was identified as Bacillus thermodenitrificans OHT-1. An 18-subunit oligonucleotide probe for 16S rRNA, labeled with fluorescein isothiocyanate, was developed for the detection of B. thermodenitrificans. Spores and vegetative cells of B. thermodenitrificans OHT-1 were detected in liquid culture and laboratory compost by whole cell hybridization using this oligonucleotide probe. The results obtained by whole cell hybridization were evaluated in growth experiments of B. thermodenitrificans OHT-1 in laboratory compost and were used to enumerate spores and vegetative cells.Key words: compost, Bacillus thermodenitrificans, 16S rRNA, whole cell hybridization.
Review / SynthèseBioaerosols from municipal and animal wastes: background and contemporary issues
Authors: Pillai Suresh D , Ricke Steven C
Published: October 2002

Global population increases, coupled with intensive animal and livestock production practices, have resulted in the generation, accumulation, and disposal of large amounts of wastes around the world. Aerosolization of microbial pathogens, endotoxins, odors, and dust particles is an inevitable consequence of the generation and handling of waste material. Bioaerosols can be a source of microbial pathogens, endotoxins, and other allergens. Given the close proximity of population centers to concentrated animal-rearing operations and municipal treatment facilities in many parts of the world, there is concern regarding the occupational and public health impacts associated with the exposure to bioaerosols from municipal and animal wastes. Major advances have been made in our understanding of bioaerosol characteristics, identifying the hazards, and identifying possible human and animal health links with aerosolized pathogens and allergens. However, significant knowledge and technology gaps still exist. These include a lack of clear understanding of the fate and transport of bioaerosols, especially within the open environment, an inability to accurately predict the health risks associated with bioaerosolized pathogens, and a lack of standardized bioaerosol sampling protocols, and efficient samplers. This review synthesizes the information related to bioaerosols and addresses the contemporary issues associated with bioaerosols from municipal and animal wastes, with a focus on pathogens.Key words: bioaerosols, pathogens, biosolids, animal wastes, health risks.
Potato-associated bacteria and their antagonistic potential towards plant-pathogenic fungi and the plant-parasitic nematodeMeloidogyne incognita(Kofoid & White) Chitwood
Authors: Krechel Annette , Faupel Annekathrin , Hallmann Johannes , Ulrich Andreas , Berg Gabriele
Published: October 2002

To study the effect of microenvironments on potato-associated bacteria, the abundance and diversity of bacteria isolated from the rhizosphere, phyllosphere, endorhiza, and endosphere of field grown potato was analyzed. Culturable bacteria were obtained after plating on R2A medium. The endophytic populations averaged 103and 105CFU/g (fresh wt.) for the endosphere and endorhiza, respectively, which were lower than those for the ectophytic microenvironments, with 105and 107CFU/g (fresh wt.) for the phyllosphere and rhizosphere, respectively. The composition and richness of bacterial species was microenvironment-dependent. The occurrence and diversity of potato-associated bacteria was additionally monitored by a cultivation-independent approach using terminal restriction fragment length polymorphism analysis of 16S rDNA. The patterns obtained revealed a high heterogeneity of community composition and suggested the existence of microenvironment-specific communities. In an approach to measure the antagonistic potential of potato-associated bacteria, a total of 440 bacteria was screened by dual testing for in vitro antagonism towards the soilborne pathogens Verticillium dahliae and Rhizoctonia solani. The proportion of isolates with antagonistic activity was highest for the rhizosphere (10%), followed by the endorhiza (9%), phyllosphere (6%), and endosphere (5%). All 33 fungal antagonists were characterized by testing their in vitro antagonistic mechanisms, including their glucanolytic, chitinolytic, pectinolytic, cellulolytic, and proteolytic activity, and by their BOX-PCR fingerprints. In addition, they were screened for their biocontrol activity against Meloidogyne incognita. Overall, nine isolates belonging to Pseudomonas and Streptomyces species were found to control both fungal pathogens and M. incognita and were therefore considered as promising biological control agents. Key words: biocontrol, antagonistic potential, plant-associated bacteria.
Identification and cloning of the bacterial nodulation specificity gene in the Sinorhizobium meliloti  Medicago laciniata symbiosis
Authors: Barran L R , Bromfield E S.P , Brown D C.W
Published: October 2002

Medicago laciniata (cut-leaf medic) is an annual medic that is highly nodulation specific, nodulating only with a restricted range of Sinorhizobium meliloti, e.g., strain 102L4, but not with most strains that nodulate Medicago sativa (alfalfa), e.g., strains RCR2011 and Rm41. Our aim was to identify and clone the S. meliloti 102L4 gene implicated in the specific nodulation of M. laciniata and to characterize the adjacent nodulation (nod) region. An 11-kb EcoRI DNA fragment from S. meliloti 102L4 was shown to complement strain RCR2011 for nodulation of M. laciniata. Nucleotide sequencing revealed that this fragment contained nodABCIJ genes whose overall arrangement was similar to those found in strains RCR2011 and Rm41, which do not nodulate M. laciniata. Data for Tn5 mutagenesis of the nodABCIJ region of strain 102L4 suggested that the nodC gene was involved in the specific nodulation of M. laciniata. Tn5 insertions in the nodIJ genes gave mutants with nodulation delay phenotypes on both M. laciniata and M. sativa. Only subclones of the 11-kb DNA fragment containing a functional nodC gene from strain 102L4 were able to complement strain RCR2011 for nodulation of M. laciniata. The practical implications of these findings are discussed in the context of the development of a specific M. sativa  S. meliloti combination that excludes competition for nodulation by bacterial competitors resident in soil.Key words: Sinorhizobium meliloti, Medicago laciniata, nodulation specificity,nod gene, nucleotide sequence.
Involvement of thehapgene (mucinase) in the survival ofVibrio choleraeO1 in association with the blue-green alga,Anabaenasp.
Authors: Islam M S , Goldar M M , Morshed M G , Khan M N.H , Islam M R , Sack R B
Published: October 2002

Mucinase is a soluble haemagglutinin protease, which may be important for the survival of Vibrio cholerae in association with mucilaginous blue-green algae (cyanobacteria). A comparative survival study was carried out with an Anabaena sp. and a wild-type V. cholerae O1 strain hap+gene (haemagglutinin-protease), together with its isogenic mutant hap (hap-deleted gene). A simple spread plate technique was followed to count culturable V. cholerae O1 on taurocholate tellurite gelatin agar plate. The fluorescent antibody technique of Kogure et al. (1979) was used for the microscopical viable count of V. cholerae O1. Polymerase chain reaction (PCR) and Southern blot hybridization were carried out to detect a lower number of viable but nonculturable (VBNC) V. cholerae O1 from the laboratory-based experiments. The wild and mutant V. cholerae O1 strains survived in culturable form for 22 and 10 days, respectively, in association with the Anabaena sp., with the difference being statistically significant (P < 0.01). The fluorescent antibody technique, PCR, and hybridization results also showed that the wild strain survived better in the VBNC state than did the mutant VBNC strain in association with an Anabaena sp. These results indicate that the enzyme mucinase may play an important role in the association and long-term survival of V. cholerae O1 with a mucilaginous blue-green alga, Anabaena sp.Key words: Vibrio cholerae, Anabaena, mucinase, reservoir.
Protein analysis in a pleomorphically deteriorated strain of the insect-pathogenic fungus Metarhizium anisopliae
Authors: Kamp Andrena M , Bidochka Michael J
Published: October 2002

Pleomorphic deterioration is a process where a fungal isolate loses the ability to produce conidia during repeated subculturing. We have previously isolated strains of the entomopathogenic fungus Metarhizium anisopliae that have irreversibly lost the ability to produce conidia and only produce mycelia when grown on agar. Gel electrophoresis was used to examine differences in intracellular protein patterns (urea-soluble proteins and urea-insoluble proteins (i.e., hydrophobins)) in conidiating and mycelial cultures of M. anisopliae. Two major proteins present in a conidiating culture and one from a mycelial culture were N-terminally sequenced but showed no homologies to known proteins. The presence of hydrophobins in conidiating and mycelial cultures was also examined, and it was shown that these proteins were abundant in conidiating cultures but not in mycelial cultures. We also used primers designed from regulatory genes involved in conidiation in Aspergillus nidulans. The amplified fragments were not homologous to A. nidulans genes.Key words: Metarhizium anisopliae, conidia, pleomorphic deterioration, protein analysis.
Inositol phosphatase activity of theEscherichia coli agp-encoded acid glucose-1-phosphatase
Authors: Cottrill Michael A , Golovan Serguei P , Phillips John P , Forsberg Cecil W
Published: October 2002

When screening an Escherichia coli gene library for myo-inositol hexakisphosphate (InsP6) phosphatases (phytases), we discovered that the agp-encoded acid glucose-1-phosphatase also possesses this activity. Purified Agp hydrolyzes glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6with pH optima, 6.5, 3.5, and 4.5, respectively, and was stable when incubated at pH values ranging from 3 to 10. Glucose-1-phosphate was hydrolyzed most efficiently at 55°C, while InsP6and p-nitrophenyl phosphate were hydrolyzed maximally at 60°C. The Agp exhibited Kmvalues of 0.39 mM, 13 mM, and 0.54 mM for the hydrolysis of glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6, respectively. High-pressure liquid chromatography (HPLC) analysis of inositol phosphate hydrolysis products of Agp demonstrated that the enzyme catalyzes the hydrolysis of phosphate from each of InsP6, D-Ins(1,2,3,4,5)P5, Ins(1,3,4,5,6)P5, and Ins(1,2,3,4,6)P5, producing D/L-Ins(1,2,4,5,6)P5, D-Ins(1,2,4,5)P4, D/L-Ins(1,4,5,6)P4and D/L-Ins(1,2,4,6)P4, respectively. These data support the contention that Agp is a 3-phosphatase. Key words: phosphatase, phytate, bacteria, inositol phosphate, phytase.
Bactericidal effect of gentamicin-induced membrane vesicles derived fromPseudomonas aeruginosaPAO1 on gram-positive bacteria
Authors: MacDonald Kelly L , Beveridge Terry J
Published: October 2002

Previous studies have shown that gentamicin-induced membrane vesicles (g-MVs) from Pseudomonas aeruginosa PAO1 possess both the antibiotic (gentamicin) and a potent peptidoglycan hydrolase (PGase; autolysin) that is effective in killing gram-negative pathogens. This present study evaluated the therapeutic potential of g-MVs against four gram-positive bacteria. Bactericidal assays and electron microscopy of thin sections revealed that Bacillus subtilis 168 and Staphylococcus aureus D2C were susceptible to killing mediated by g-MVs, Listeria monocytogenes ATCC 19113 was slightly susceptible, whereas Enterococcus hirae ATCC 9790 was unaffected. g-MVs were generally more effective against the bacteria than was soluble gentamicin, suggesting they could have more killing power than natural membrane vesicles containing no antibiotic. Electron microscopy and hydrophobic interaction chromatography showed that more membrane vesicles (MVs) initially attached to B. subtilis (hydrophilic) than to predominantly hydrophobic E. hirae, L. monocytogenes, and S. aureus. Zymograms containing murein sacculi as an enzyme substrate illustrated that all organisms except E. hirae were sensitive to the 26-kDa autolysin to varying degrees. Peptidoglycan O-acetylation did not influence susceptibility to MV-mediated lysis. Though not universally effective, the g-MV delivery system remains a promising therapeutic alternative for specific gram-positive infections.Key words: gram-negative membrane vesicles, gentamicin, autolysin.
Regulation of Sinorhizobium meliloti 1021 rrnA-reporter gene fusions in response to cold shock
Authors: Gustafson Ann M , O'Connell Kevin P , Thomashow Michael F
Published: October 2002

We previously reported that mutants of Sinorhizobium meliloti 1021 carrying luxAB insertions in each of the three 16S rRNA genes exhibited a dramatic ([Formula: see text]28-fold) increase in luminescence following a temperature downshift from 30 to 15°C. These results raised the possibility that the rRNA operons (rrn) of S. meliloti were cold shock loci. In testing this possibility, we found that fusion of the S. meliloti 1021 rrnA promoter to two different reporter genes, luxAB and uidA, resulted in hybrid genes that were transiently upregulated (as measured by transcript accumulation) about four- to sixfold in response to a temperature downshift. These results are consistent with the hypothesis that the rrn promoters are transiently upregulated in response to cold shock. However, much of the apparent cold shock regulation of the initial luxAB insertions was due to an unexpected mechanism: an apparent temperature-dependent inhibition of translation. Specifically, the rrnA sequences from +1 to +172 (relative to the start of transcription) were found to greatly decrease the ability of S. meliloti to translate hybrid rrnluxAB transcripts into active protein at 30°C. This effect, however, was largely eliminated at 15°C. Possible mechanisms for the apparent transient increase in rrnA promoter activity and temperature-dependent inhibition of translation are discussed.Key words: rhizobium, gene expression, luciferase.
Genetic relatedness of Trichoderma sect. Pachybasium species based on molecular approaches
Authors: Lee Ching-Fu , Hseu Tzong-Hsiung
Published: October 2002

Molecular approaches including internal transcribed spacer (ITS) sequences of ribosomal DNA, universal primer polymerase chain reaction (UP-PCR) fingerprinting, and DNADNA hybridization were used to study the genetic relatedness of species within Trichoderma sect. Pachybasium. In the analysis of ITS and 5.8S sequences of ribosomal DNA, parsimony analysis demonstrated that forty-one strains were distributed into five main groups supported by high bootstrap values. The species of Trichoderma sect. Pachybasium were clustered into groups I, II, and IV, with the strains of Trichoderma fasculatum and Trichoderma strictipile forming a separate branch, an independent group V. Some species within each group showed nearly identical sequence differences (fewer than 13 bp). UP-PCR and DNADNA hybridization were further used to clarify the genetic relatedness of these species with highly similar ITS sequences. Highly similar or identical UP-PCR profiles and high values of DNA complementarity (>70%) were observed among some species, Trichoderma hamatum and Trichoderma pubescens; Trichoderma croceum,Trichoderma polysporum and Trichoderma album; Trichoderma crassum and Trichoderma flavofuscum; and Trichoderma strictipile and Trichoderma fasciculatum. Although every species can be differentiated morphologically, the species showed highly similar molecular characteristics in the above cases, indicating that they could be conspecific. However, in some cases (Trichoderma longipile, T. crassum and T. flavofuscum; Trichoderma fertile and Trichoderma minutisporum; Trichoderma tomentosum, Trichoderma inhamatum and Trichoderma harzianum) there were discriminative patterns of UP-PCR and (or) low levels (<50%) of DNADNA hybridization; even their ITS sequences were similar, suggesting a closely phylogenetic relationship.Key words: fingerprinting, ITS sequencing, DNADNA hybridization, taxonomy, Trichoderma.
Development of a selective myclobutanil agar (MBA) medium for the isolation of Fusarium species from asparagus fields
Authors: Vujanovic Vladimir , Hamel Chantal , Jabaji-Hare Suha , St-Arnaud Marc
Published: October 2002

A new selective myclobutanil agar medium for the detection of Fusarium species is proposed. Ten media formulations based on various selective agents (pentachloronitrobenzene (PCNB), Rose Bengal, malachite green, sodium hypochlorite, captan, benomyl, chlorotalonil, myclobutanil, thiram, and cupric sulfate) were compared. First, mycelium growth and colony appearance of Alternaria alternata, Aspergillus flavus, Cladosporium cladosporioides, Epicoccum nigrum,Fusarium sp., Fusarium solani, Fusarium moniliforme, Fusarium oxysporum f.sp. dianthi, Penicillium sp., and Trichoderma viride isolates were compared. Second, the ability of the different media to isolate and enumerate fusaria from asparagus fields was evaluated. The myclobutanil-based medium showed the highest selectivity to Fusarium spp. growth but required a slightly longer incubation time (>5 d) than peptonepentachloronitrobenzene-based agar (PPA) (< 5 d). PPA allowed a faster fusaria growth but also permited the growth of other moulds. The other media were less selective and did not allow to isolate fusaria or to differenciate them from other growing fungi.Key words: selective medium, myclobutanil, Fusarium, soil, Asparagus.
Solution structure of a DNA duplex containing a formamide-adenine base pair
Authors: Maufrais Corinne , Boulard Yves
Published: October 2002

The N-(2-deoxy-beta-D-erythro-pentofuranosyl) formamide residue results from a ring fragmentation product of thymine or cytosine. The presence of a formamide-adenine base pair in the sequence 5'd(AGGAACCACG).d(CGTGGFTCCT) has been studied by 1H and 31P nuclear magnetic resonance (NMR) and molecular dynamics. There are two possible isomers for the formamide side chain, either cis or trans. For each isomer, we observed an equilibrium in solution between two forms. First, a species where the formamide is intrahelical and paired with the facing adenine. For the cis isomer, the formamide is in a syn conformation and two hydrogen bonds with adenine are formed. The trans isomer is in an anti conformation and a single hydrogen bond is observed. In the second form, whatever the isomer, the formamide is rejected outside the helix, whereas the adenine remains inside. Key words: DNA structure, mutagenesis, NMR, molecular dynamics, formamide.
Radiation could induce p53-independent and cell cycle - unrelated apoptosis in 5-fluorouracil radiosensitized head and neck carcinoma cells
Authors: Didelot C , Mirjolet J F , Barberi-Heyob M , Ramacci C , Merlin J L
Published: October 2002

The effect of chemoresistance induction in radiosensitivity and cellular behavior after irradiation remains misunderstood. This study was designed to understand the relationship between radiation-induced cell cycle arrest, apoptosis, and radiosensitivity in KB cell line and KB3 subline selected after 5-fluorouracil (5FU) exposure. Exposure of KB cells to 5FU led to an increase in radiosensitivity. G2/M cell cycle arrest was observed in the two cell lines after irradiation. The radioresistant KB cell line reached the maximum arrest two hours before KB3. The cellular exit from this arrest was found to be related to the wild type p53 protein expression induction. After irradiation, only KB3 cell line underwent apoptosis. This apoptosis induction seemed to be independent of G2/M arrest exit, which was carried out later. The difference in radiosensitivity between KB and KB3 subline may result therefore from both a difference in apoptosis induction and a difference in G2/M arrest maximum duration. Moreover, 5FU exposure has led to an increase in constitutive p53 protein expression, which may be associated with an increase in basal apoptosis cell fraction. Given the existing correlation between radiosensitivity and the percentage of basal apoptosis, the constitutive p53 protein expression may be related to intrinsic radiosensitivity in our cellular model.Key words: cellular radiosensitivity, G2/M arrest, p53, apoptosis.
Blood-brain barrier permeability after gamma whole-body irradiation: an in vivo microdialysis study
Authors: Diserbo Michel , Agin Arnaud , Lamproglou Ioannis , Mauris Jérôme , Staali Fatiha , Multon Eric , Amourette Christine
Published: October 2002

The effects of total-body irradiation on the permeability of rat striatal blood-brain barrier (BBB) to [3H]alpha-aminoisobutyric acid (AIBA) and [14C]sucrose were investigated using the microdialysis technique. Seven days, 3 and 6 weeks, and 3, 5, and 8 months after gamma exposure at a dose of 4.5 Gy, no modification of the permeability to both [3H]AIBA and [14C]sucrose was observed. But, in the course of the initial syndrome, we observed a significant but transient increase in the BBB permeability to the two markers between 3 and 17 h after exposure. A secondary transient "opening" of the BBB to [14C]sucrose was noticed about 28 h following irradiation without the corresponding increase in BBB permeability to [3H]AIBA. On the contrary, the transport of [3H]AIBA through the BBB was decreased between 33 and 47 h postradiation. In conclusion, our experiments showed early modifications of BBB permeability after a moderate-dose whole-body exposure. Confirmation of these results with other tracers, in another experimental model or in humans, would have clinical applications for designing appropriate pharmacotherapy in radiotherapy and treatment of accidental overexposure.Key words: radiation effects, central nervous system, blood-brain barrier, moderate dose, microdialysis.
Effet des rayonnements ionisants sur les cellules souches et progéniteurs hématopoïétiques: place de l'apoptose et intérêt thérapeutique potentiel des traitements antiapoptotiques
Authors: Drouet M , Mourcin F , Grenier N , Mayol J F , Leroux V , Sotto J J , Hérodin F
Published: October 2002

Bone marrow aplasia observed following ionizing radiation exposure (Total Body Irradiation; gamma dose range: 210 Gy) is a result, in particular, of the radiation-induced (RI) apoptosis in hematopoietic stem and progenitor cells (HSPC). We have previously shown in a baboon model of mobilized peripheral blood CD34+cell irradiation in vitro that RI apoptosis in HSPC was an early event, mostly occurring within the first 24 hours, which involves the CD95 Fas pathway. Apoptosis may be significantly reduced with a combination of 4 cytokines (4F): Stem Cell Factor (SCF), FLT-3 Ligand (FL), thrombopoietin (TPO), and interleukin-3 (IL-3), each at 50 ng·mL1(15% survival versus <3% untreated cells, 24 h post-irradiation at 2.5 Gy). In this study we show that addition of TNF-alpha(800 IU/ml) induces an increase in 4F efficacy in terms of cell survival 24 h after incubation (26% survival after 24 h irradiation exposure at 2.5 Gy) and amplification (k) of CD34+cells after 6 days in a serum free culture medium (SFM) (kCD34+= 4.3 and 6.3 respectively for 4F and successive 4F + TNF-alpha/ 4F treatments). In addition, the 4F combination allows culture on pre-established allogenic irradiated stromal cells in vitro at 4 Gy (kCD34+= 4.5). Overall this study suggests (i) the potential therapeutic interest for an early administration of anti-apoptotic cytokines with or without hematopoiesis inhibitors (emergency cytokine therapy) and (ii) the feasibility in the accidentally irradiated individual, of autologous cell therapy based on ex vivo expansion in order to perform autograft of residual HSPC collected after the accident.Key words: apoptosis, cytokine, hematopoiesis, irradiation, bone marrow aplasia.[Journal translation]
Induction of apoptosis by high linear energy transfer radiation: role of p53
Authors: Coelho D , Fischer B , Holl V , Dufour P , Denis J M , Gueulette J , Bergerat J P , Bischoff P
Published: October 2002

The involvement of the tumor suppressor p53 gene in the sensitivity of many cell types towards low linear energy transfer (LET) radiation is now well established. However, little information is available on the relationship between p53 status of tumor cells and their ability to undergo apoptosis following exposure to high-LET radiation. Here we present the results of experiments carried out with the human lymphoblastoid cell line TK6 and its p53 knock-out counterpart NH32. Cells were irradiated at doses ranging from 0.25 to 8 Gy with fast neutrons (65 MeV), carbon ions (95 MeV/nucleon), and X rays (15 MV). For both cell lines, the occurrence of apoptosis, determined by the quantification of hypodiploid particles as well as the activation of several caspases, was compared with their sensitivity towards high-LET radiation. Results indicate that p53 is involved in the response of TK6 cells to fast neutrons and carbon ions, as measured by cell proliferation and occurrence of apoptosis. However, p53-deficient cells are still able to undergo apoptosis following irradiation. This suggests that heavy ions and fast neutrons induce cellular damage that is not under the control of p53. The involvement of executioner caspases in high-LET radiation induced apoptosis was also evaluated by use of specific inhibitors.Key words: fast neutrons, carbon ions, apoptosis, p53, lymphoblastoid cell line.
Mesure de paramètres spécifiques pour le calcul de dose après inhalation d'aérosols renfermant des éléments transuraniens
Authors: Gall B Ramounet-Le , Fritsch P , Abram M C , Rateau G , Grillon G , Guillet K , Baude S , Bérard P , Ansoborlo E , Delforge J
Published: October 2002

A review on specific parameter measurements to calculate doses per unit of incorporation according to recommendations of the International Commission of Radiological Protection has been performed for inhaled actinide oxides. Alpha activity distribution of the particles can be obtained by autoradiography analysis using aerosol sampling filters at the work places. This allows us to characterize granulometric parameters of "pure" actinide oxides, but complementary analysis by scanning electron microscopy is needed for complex aerosols. Dissolution parameters with their standard deviation are obtained after rat inhalation exposure, taking into account both mechanical lung clearance and actinide transfer to the blood estimated from bone retention. In vitro experiments suggest that the slow dissolution rate might decrease as a function of time following exposure. Dose calculation software packages have been developed to take into account granulometry and dissolution parameters as well as specific physiological parameters of exposed individuals. In the case of poorly soluble actinide oxides, granulometry and physiology appear as the main parameters controlling dose value, whereas dissolution only alters dose distribution. Validation of these software packages are in progress.Key words: actinide oxides, dosimetry, inhalation.
Autologous cell therapy as a new approach to treatment of radiation-induced bone marrow aplasia: preliminary study in a baboon model
Authors: Hérodin F , Drouet M
Published: October 2002

The sparing of viable hematopoietic stem and progenitor cells located in underexposed bone marrow territories associated with the relative radioresistance of certain stem cell populations is the rationale for autologous cell therapy consisting of ex vivo expansion of residual cells after collection postirradiation. The feasibility of this treatment mainly depends on time constraints and hematopoietic cell threshold. We showed in this study that in the absence of early-acting mobilizing agent administration, subliminar amounts of CD34+ cells can be collected (1 × 106 CD34+ cells/100 mL bone marrow or for 1 L apheresis) from 6-Gy gamma globally irradiated baboons. Residual CD34+ cells were successfully expanded in serum-free medium in the presence of antiapoptotic cytokine combination (stem cell factor + FLT-3 ligand + thrombopoietin + interleukin 3, 50 ng/mL each, i.e., 4F): KCD34+ = ×2.8 and ×13.7 (n = 2). Moreover, we demonstrated the short-term neutrophil engraftment potential of a low-size mixed expanded graft (1.5 × 106 final CD34+cells/kg) issued from the coculture of unirradiated (20%) and 2.5-Gy in vitro irradiated (80%) CD34+ cells on an allogeneic stromal cell layer in the presence of 4F. Further preclinical research needs to be performed to clearly establish this therapeutic approach that could be optimized by the early administration of antiapoptotic cytokines.Key words: ex vivo expansion, cytokine, cell therapy, bone marrow aplasia, irradiation, animal model.
Inhibition of ultraviolet B (UVB) induced apoptosis in A431 cells by mimosine is not dependent on cell cycle arrest
Authors: Cliche D O , Girouard S , Bissonnette N , Hunting D J
Published: October 2002

Ultraviolet (UV) radiation is a strong apoptotic trigger in many cell types. We have previously reported that a plant amino acid, mimosine (beta-[N-(3-hydroxy-4-pyridone)]-alpha-aminopropionic acid), with a well-known reversible G1 cell cycle arrest activity can inhibit apoptosis induced by UV irradiation and RNA polymerase II blockage in human A431 cells. Here, apoptosis was measured with a fluorimetric caspase activation assay. Interestingly, the protective state was effective up to 24 h following removal of mimosine from the culture medium while cells were progressing in the cell cycle. Our results demonstrate that the protective effect of mimosine against UV-induced apoptosis can be dissociated from its G1 cell-cycle arrest activity.Key words: mimosine, apoptosis, cell cycle, A431 cells, caspase activation assay.
Mécanisme moléculaire de l'effet protecteur de la vitamine E dans l'athérosclérose
Authors: Khalil Abdelouahed
Published: October 2002

Oxidation of low-density lipoproteins constitutes the first step of a very complex process leading to atherosclerosis. Vitamin E, and principally alpha-tocopherol, is considered as the principal inhibitor of lipid peroxidation. Some studies showed the beneficial role of vitamin E in the prevention and reduction of atherosclerosis and its associated pathologies. However, other in vitro studies advance a prooxidant role of vitamin E. The results of the epidemiologic studies are difficult to generalize without taking account of the clinical randomized tests. In this work, we reviewed the principal studies devoted to the role of vitamin E and discussed the assumption of a prooxidant effect of this molecule.Key words: vitamin E, low-density lipoproteins (LDL), lipid peroxidation, cardio-vascular diseases.
Influence de l'âge sur les troubles cognitifs radio-induits: études expérimentales avec irradiation cérébrale de 30 Gy en 10 séances et 12 jours chez le rat Wistar de 1 1/2, 4 et 18 mois
Authors: Lamproglou I , Baillet F , Boisserie G , Mazeron J J , Delattre J Y
Published: October 2002

The objective of this study was to determine the influence of age on the learning and memory dysfunction induced by cranial radiation in the male Wistar rat. Ninety-six 45-day-old, 70 4-month-old, and 78 18-month-old male rats were divided in two equal groups: (i) irradiated and (ii) control. A course of whole-brain radiation therapy (30 Gy in 10 fractions over 12 days) was administered to the irradiated group, while the control group received sham irradiation. Sequential behavioral studies including one and two-way avoidance tests were undertaken before and after the 7 months following radiation. The results suggest that radiation induced progressive and irreversible memory dysfunction in elderly (18-month-old) rats, but this effect was partial or almost reversible in the 4-month-old and 45-day-old rats, respectively. In return, the learning dysfunction was age non-dependent despite the fact that is occurs more rapidly in the young (45 days, 4 months) rats.Key words: radiation therapy, brain, cognition, rat.