Motile chemosensory behaviour of phagotrophic protists: mechanisms for and efficiency in congregating at food patches

Phagotrophic protists are capable of congregating at point sources of food within a few minutes, from distances of up to several cm in the case of ciliates, or several mm in the case of microflagellates. This is exemplified by four ciliate species and a heterotrophic flagellate. Congregation is accomplished by the combined effect of more than one type of chemosensory motile behaviour including "kinetic responses", "temporal-gradient sensing", and "helical klinotaxis". The results are discussed in terms of microscale patchiness in nature.     

Bioluminescent tracing of a Yersinia pestis pCD1+-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice

Yersinia pestis has evolved from Yersinia pseudotuberculosis serotype O:1b. A typical Y. pestis contains three plasmids: pCD1, pMT1 and pPCP1. However, some isolates only harbor pCD1 (pCD1⁺-mutant). Y. pestis and Y. pseudotuberculosis share a common plasmid (pCD1 or pYV), but little is known about whether Y. pseudotuberculosis exhibited plague-inducing potential before it was evolved into Y. pestis. Here, the luxCDABE::Tn5::kan was integrated into the chromosome of the pCD1⁺-mutant, Y. pseudotuberculosis or Escherichia coli K12 to construct stable bioluminescent strains for investigation of their dissemination in mice by bioluminescence imaging technology. After subcutaneous infection, the pCD1⁺-mutant entered the lymph nodes, followed by the liver and spleen, and, subsequently, the lungs, causing pathological changes in these organs. Y. pseudotuberculosis entered the lymph nodes, but not the liver, spleen and lungs. It also resided in the lymph nodes for several days, but did not cause lymphadenitis or pathological lesions. By contrast, E. coli K12-lux was not isolatable from mouse lymph nodes, liver, spleen and lungs. These results indicate that the pCD1⁺-mutant can cause typical bubonic and pneumonic plague-like diseases, and Y. pestis has inherited lymphoid tissue tropism from its ancestor rather than acquiring these properties independently.     

Validation of a simple liquid chromatography-tandem mass spectrometric method for the determination of propiverine hydrochloride and its N-oxide metabolite in human plasma

A simple high-performance liquid chromatography (HPLC)-tandem mass spectrometric method has been developed for determination of propiverine hydrochloride and its metabolite, propiverine N-oxide (M-1) in human plasma using stable isotopes, propiverine hydrochloride-d10 and M-1-d10, as internal standards. The analytes were extracted with dichloromethane from 0.2 ml of plasma in neutral condition (pH 7.0) and separated by HPLC on a C18 reversed-phase column using methanol-1% acetic acid (50:50) as a mobile phase, and detected using positive electrospray ionization in selected reaction monitoring (SRM) mode. The method was validated over a concentration range of 2-500 ng/ml for propiverine hydrochloride and 4-1000 ng/ml for M-1 using 0.2 ml of human plasma per assay. The method developed was successfully applied to analysis of propiverine hydrochloride and M-1 in clinical studies.     

Keystone Symposia on Molecular and Cellular Biology: ‘The molecular basis of cancer’: Organizers: Carol L. Prives and George F. Vande Woude,Taos, NM, 15–21 March 1999

The Keystone Symposium on the Molecular Basis of Cancer was an excellent meeting, which stimulated the exchange of a great deal of information. This report was prepared to organize some of the results that provided new insights into the regulation of cell proliferation and apoptosis. We were unable to report on all of the talks and posters due mostly to our limited capacity to absorb and digest the large amount of results presented at the meeting. We apologize to those whose results were not covered in this report.     

Characterization of p80, a novel nuclear and cytoplasmic protein in dinoflagellates.

The presence of myosin in dinoflagellates was tested using an anti-Acanthamoeba castellanii myosin II polyclonal antibody on the heterotrophic dinoflagellate Crypthecodinium cohnii Seligo. Western blots revealed the presence of a unique band of 80 kDa in total protein extracts and after immunoprecipitation. Expression of this 80 kDa protein appeared constant during the different phases of the cell cycle. In protein extracts from various other dinoflagellates, this 80 kDa protein was detected only in the autotrophic species Prorocentrum micans Ehr. Screening of a C. cohnii cDNA expression library with this antibody revealed a cDNA coding for an amino acid sequence without homology in the databases. However, particular regions were detected: - a polyglutamine repeat domain in the N-terminal part of the protein, - four peptide sequences associated with GTP-binding sites, - a sequence with slight homology to the rod tail of Caenorhabditis elegans myosin II, -a sequence with homology to a human kinesin motor domain. Immunocytolocalization performed on C. cohnii thin sections with a polyclonal antibody raised against the recombinant protein showed p80 to be present both within the nucleus and in the cytoplasm. Labelling was widespread in the nucleoplasm and more concentrated at the periphery of the permanently condensed chromosomes. In the cytoplasm, labelling appeared in a punctate region close to the nucleus and in the flagellum. Potential functions of this novel protein are discussed.     

Taxonomic update of Clitocybula sensu lato with a new generic classification

The taxonomy and phylogeny of the hydropoid clade (genera Clitocybula s.l., Megacollybia, Leucoinocybe gen. nov., Hydropus, Trogia, Gerronema, Porotheleum and Lignomphalia gen. nov.) in Europe is studied using morphological and molecular approaches; the first three genera in detail including all known European species. Only two European species remain in Clitocybula s.str., Clitocybula lacerata and Clitocybula familia. The European C. lacerata is a species complex which should be treated as C. lacerata agg. at the current state of knowledge. A neotype originating from type area was designated to fix the application of the name. The presence of American species Clitocybula abundans in Europe is insufficiently proved. “Clitocybula dryadicola ? belongs to the genus Hydropus, and Clitocybula tilieti has an unclear systematic position. The results showed that Megacollybia and Leucoinocybe represent independent genera separated from Clitocybula. The genus Leucoinocybe is validly published with two European species, Leucoinocybe lenta and Leucoinocybe taniae. “Clitocybula flavoaurantia” proved to be conspecific with the latter species. The genus Lignomphalia is published for “Pseudoomphalina lignicola”, a lignicolous omphalinoid species. The Indian “Clitocybula sulcata” is transferred to Leucoinocybe and “Clitocybula atrialba” to Gerronema. The first European records of Megacollybia marginata are published.     

Lipase of Pseudomonas cepacia for biotechnological purposes: purification,crystallization and characterization

Commercial lipase (triacylglycerol lipase, EC 3.1.1.3) of Pseudomonas cepacia (Amano) has been purified to homogeneity by a single chromatography on phenyl Sepharose. The eluted lipase crystallized spontaneously at 4°C in the eluent, containing 58–69% 2-propanol. The yield of the lipase was 87–100% and the specific activity during the hydrolysis of triolein 5800 U/mg protein. This protein has a molecular weight of 34.1 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its purity was determined by SDS-Page and capillary zone electrophoresis to be ≥ 99%. Immobilization on Sepharose increased its stability in organic solvents. This lipase of P. cepacia differs from that of other Pseudomonas strains in respect of substrate specificity and during crystallization. It exhibits a high stability in organic solvents and supercritical carbon dioxide.     

Examination into the taxonomic position of Bacillus thermotolerans Yang et al., 2013, proposal for its reclassification into a new genus and species Quasibacillus thermotolerans gen. nov., comb. nov. and reclassification of B. encimensis Dastager et al., 2015 as a later heterotypic synonym of B. badius

Two novel Gram-staining positive, rod-shaped, moderately halotolerant, endospore forming bacterial strains 5.5LF 38TD and 5.5LF 48TD were isolated and taxonomically characterized from a landfill in Chandigarh, India. The analysis of 16S rRNA gene sequences of the strains confirmed their closest identity to Bacillus thermotolerans SgZ-8T with 99.9% sequence similarity. A comparative phylogenetic analysis of strains 5.5LF 38TD, 5.5LF 48TD and B. thermotolerans SgZ-8^T confirmed their separation into a novel genus with B. badius and genus Domibacillus as the closest phylogenetic relatives. The major fatty acids of the strains are iso-C_15:0 and iso-C_16:0 and MK-7 is the only quinone. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The digital DNA-DNA hybridization (DDH) and ortho average nucleotide identity (ANI) values calculated through whole genome sequences indicated that the three strains showed low relatedness with their phylogenetic neighbours. Based on evidences from phylogenomic analyses and polyphasic taxonomic characterization we propose reclassification of the species B. thermotolerans into a novel genus named Quasibacillus thermotolerans gen. nov., comb. nov with the type strain SgZ-8^T (= CCTCC AB2012108^T = KACC 16706^T). Further our analyses also revealed that B. encimensis SGD-V-25^T is a later heterotypic synonym of Bacillus badius DSM 23^T.     

Role of p53 and mismatch repair in PhIP-induced perturbations of the cell cycle

Heterocyclic amines, found ubiquitously in our diet, are carcinogenic and mutagenic. Among this class of compounds, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) is the most abundant. To further understand the carcinogenesis of this compound, we studied the effects of PhIP on the progression of human lymphoblastoid cells through the cell-cycle. Cells differing in p53 or mismatch repair status were used to evaluate the role of those proteins. Following PhIP-treatment, a dose and time-dependent accumulation of p53 was found in cells containing functional p53. The augmentation of the p53 protein, accompanied by increases in p21-WAF1, confirms that the p53 is activated. The increase in p53 was independent of the mismatch repair status of the cells. Perturbations in the cell-cycle were also observed. Twenty-four hours after PhIP treatment, the activation of the G2-M checkpoint was evident. Functional p53 and mismatch repair were not required for the PhIP-induced G2-M arrest. The G2-M arrests were reversible and are interpreted as necessary for the repair of the PhIP-DNA lesions. Under treatment conditions where less than 5% of the cells survived, the G2-M arrests were absent.     

Preferential infectivity of entomopathogenic nematodes in an envenomed host

Entomopathogenic nematodes and parasitoid wasps are used as biological control agents for management of insect pests such as the Indian meal moth, Plodia interpunctella. The parasitoid wasp Habrobracon hebetor injects a paralytic venom into P. interpunctella larvae before laying eggs. A previous study reported that the entomopathogenic nematode Heterorhabditis indica preferentially infects P. interpunctella that have been envenomed by H. hebetor while results in this study showed a similar preference by the entomopathogenic nematode, Steinernema glaseri. We therefore tested four hypotheses for why nematode infection rates are higher in envenomed hosts: (1) elevated CO₂ emission from envenomed hosts attracts nematodes, (2) paralysis prevents hosts from escaping nematodes, (3) volatile chemicals emitted from envenomed hosts attract nematodes and increase infection, and (4) reduced immune defenses in envenomed hosts increase nematode survival. Results showed that envenomed P. interpunctella larvae emitted lower amounts of CO₂ than non-envenomed larvae. Physical immobilization of P. interpunctella larvae did not increase infection rates by S. glaseri but did increase infection rates by H. indica. Emissions from envenomed hosts were collected and analyzed by thermal desorption gas chromatography/mass spectrometry. The most abundant compound, 3-methyl-3-buten-1-ol, was found to be an effective cue for S. glaseri attraction and infection but was not an effective stimulus for H. indica. Envenomed P. interpunctella exhibited a stronger immune response toward nematodes than non-envenomed hosts. Altogether, we conclude that different mechanisms underlie preferential infection in the two nematode species: host immobilization for H. indica and chemical cues for S. glaseri.     

Suitability of dried herbarium specimens for NMR metabolomics of mushrooms. A comparison of four species of the genera Kuehneromyces and Hypholoma (Strophariaceae)

Herbarium specimens are a treasure trove for biochemical studies. However, this implies understanding of the chemical changes during the drying and storage of the specimen. We compared herbarium specimens at different ages and fresh samples of four mushroom species (Kuehneromyces mutabilis, Hypholoma capnoides, Kuehneromyces lignicola, Hypholoma fasciculare) of two genera in the family Strophariaceae by using proton nuclear magnetic resonance (¹H NMR) spectroscopy combined with principal component analysis (PCA). 25 metabolites were identified. No significant alterations were found between herbarium samples at different ages, suggesting that they are stable enough for comparative studies. The most dominant differences between fresh and herbarium samples was that sugars such as α-α-trehalose, and fumaric and malic acids were more abundant in fresh fungi. Total contents of fatty and amino acids, uracil and γ-aminobutyric acid (GABA) were higher in herbarium specimens. In addition, pyroglutamic acid was observed only in Kuehneromyces mutabilis and fasciculic acid E in Hypholomafasciculare. Hence, based on results of the studied taxa, we conclude that NMR metabolomics can be used for both fresh and dried mushrooms when such alterations are properly addressed.     

Comparison of aquatic hyphomycetes communities between lotic and lentic environments in the Atlantic rain forest of Pernambuco,Northeast Brazil

Riparian forests are important to aquatic ecosystems and produce large quantities of organic matter that are recycled by the microbial community that includes microscopic fungi. The aim of this study was to unveil and compare the diversity of aquatic hyphomycetes associated to submerged leaf litter of tropical lotic and lentic environments in the Atlantic Forest of Northeast Brazil. Six sampling events were carried out in six points of two study areas: Biological Reserve “Mata da Chuva” (MC) and Environmental Protection Area “Lagoa da Mata” (LM), in Pernambuco, Brazil. Twenty three taxa of hyphomycetes were identified resulting in 87 occurrences. In the lake LM, 13 taxa of hyphomycetes were identified with 34 occurrences and in the MC (stream), 20 taxa with 53 occurrences. Ten species were common to both areas. Diversity indices and fungal biomass (ergosterol) were mostly higher in the lotic system. The fungal community analysis did not show any structure regarding sampling periods or sampling points within an area, however the two areas are different. Although the turbulence of the water is considered important for the development of these aquatic fungi, it is possible to find a diverse community of hyphomycetes and considerable fungal biomass in the lentic environment.     

Life history and functional capacity of the microbiome are altered in beta-cypermethrin-resistant cockroaches

Cockroaches are widely perceived to evolve resistance to insecticides. Over-expression of a resistance-conferring gene can be costly and may require energy and resource reallocation for metabolic and developmental processes. To evaluate whether changes in the composition of gut microbiota in Blattella germanica affected its resistance evolution to beta-cypermethrin and to determine the role of gut microbiota in host growth and development, we studied the relationship between insecticide resistance and the diversity and genetic content of gut microbiota in cockroaches. Results suggest beta-cypermethrin-resistant cockroaches (R strain) exhibited a delayed development period and reduced adult longevity compared with susceptible cockroaches (S strain). Based on 16S rRNA gene sequencing and community metagenomics, we found that the relative abundance of Lactobacillus and Acetobacteraceae were significantly lower in the R strain compared with the S strain in the foregut and midgut of both strains. Functional annotation of Kyoto Encyclopedia of Genes and Genomes (KEGG) modules of midgut genes in the two strains revealed that 10.6% were involved in metabolism, while the relative abundance in the R strain was 7.4%. Unigenes were also translated into amino acid sequences and assigned to protein families based on hits to the Carbohydrate-Active enzymes (CAZy) database. This process identified the glycoside hydrolases, glycosyl transferases and carbohydrate-binding modules of the S strain as all being significantly higher in diversity than those in the R strain. Overall, we conclude that fitness-related costs increased in the resistant strain of cockroaches compared with the susceptible strain, and the variation in insect gut microbiota, especially those related to growth and development, was an important influencing factor.