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PubMed2004

Comparison of 16S rRNA gene sequences of genus Methanobrevibacter.

BACKGROUND: The phylogeny of the genus Methanobrevibacter was established almost 25 years ago on the basis of the similarities of the 16S rRNA oligonucleotide catalogs. Since then, many 16S rRNA gene sequences of newly isolated strains or clones representing the genus Methanobrevibacter have been deposited. We tried to reorganize the 16S rRNA gene sequences of this genus and revise the taxonomic affiliation of the isolates and clones representing the genus Methanobrevibacter. RESULTS: The phylogenetic analysis of the genus based on 786 bp aligned region from fifty-four representative sequences of the 120 available sequences for the genus revealed seven multi-member groups namely, Ruminantium, Smithii, Woesei, Curvatus, Arboriphilicus, Filiformis, and the Termite gut symbionts along with three separate lineages represented by Mbr. wolinii, Mbr. acididurans, and termite gut flagellate symbiont LHD12. The cophenetic correlation coefficient, a test for the ultrametric properties of the 16S rRNA gene sequences used for the tree was found to be 0.913 indicating the high degree of goodness of fit of the tree topology. A significant relationship was found between the 16S rRNA sequence similarity (S) and the extent of DNA hybridization (D) for the genus with the correlation coefficient (r) for logD and logS, and for [ln(-lnD) and ln(-lnS)] being 0.73 and 0.796 respectively. Our analysis revealed that for this genus, when S = 0.984, D would be <70% at least 99% of the times, and with 70% D as the species "cutoff", any 16S rRNA gene sequence showing <98% sequence similarity can be considered as a separate species. In addition, we deduced group specific signature positions that have remained conserved in evolution of the genus. CONCLUSIONS: A very significant relationship between D and S was found to exist for the genus Methanobrevibacter, implying that it is possible to predict D from S with a known precision for the genus. We propose to include the termite gut flagellate symbiont LHD12, the methanogenic endosymbionts of the ciliate Nyctotherus ovalis, and rat feces isolate RT reported earlier, as separate species of the genus Methanobrevibacter.

Animals↗
PubMed2005

LmxMPK4, a mitogen-activated protein (MAP) kinase homologue essential for promastigotes and amastigotes of Leishmania mexicana.

BACKGROUND: Leishmania parasites undergo profound morphological and biochemical changes while passing through their life cycle. Protein kinases have been shown to be involved in the differentiation from the extracellular flagellated promastigotes to the intracellular "non-flagellated" amastigotes and vice versa. Moreover, these enzymes are likely involved in the regulation of the proliferation of the different life stages. RESULTS: Here, we characterize LmxMPK4, a mitogen-activated protein (MAP) kinase homologue from Leishmania mexicana. The kinase reveals all sequence motifs for classification as a MAP kinase. LmxMPK4 proved to be active as a recombinant protein. The kinase is expressed in promastigotes and amastigotes. It was impossible to generate homozygous gene deletion mutants for LmxMPK4 in promastigotes. Moreover, amastigotes bearing only an episomal copy of the gene stably retained LmxMPK4 over a prolonged period without antibiotic pressure in infected mice. CONCLUSION: LmxMPK4 is essential for promastigotes and amastigotes of Leishmania. It shows significant amino acid sequence divergence to mammalian MAP kinases. Thus, LmxMPK4 is a promising new drug target.

Journal Article↗
PubMed2006

Rooting the tree of life by transition analyses.

BACKGROUND: Despite great advances in clarifying the family tree of life, it is still not agreed where its root is or what properties the most ancient cells possessed--the most difficult problems in phylogeny. Protein paralogue trees can theoretically place the root, but are contradictory because of tree-reconstruction artefacts or poor resolution; ribosome-related and DNA-handling enzymes suggested one between neomura (eukaryotes plus archaebacteria) and eubacteria, whereas metabolic enzymes often place it within eubacteria but in contradictory places. Palaeontology shows that eubacteria are much more ancient than eukaryotes, and, together with phylogenetic evidence that archaebacteria are sisters not ancestral to eukaryotes, implies that the root is not within the neomura. Transition analysis, involving comparative/developmental and selective arguments, can polarize major transitions and thereby systematically exclude the root from major clades possessing derived characters and thus locate it; previously the 20 shared neomuran characters were thus argued to be derived, but whether the root was within eubacteria or between them and archaebacteria remained controversial. RESULTS: I analyze 13 major transitions within eubacteria, showing how they can all be congruently polarized. I infer the first fully resolved prokaryote tree, with a basal stem comprising the new infrakingdom Glidobacteria (Chlorobacteria, Hadobacteria, Cyanobacteria), which is entirely non-flagellate and probably ancestrally had gliding motility, and two derived branches (Gracilicutes and Unibacteria/Eurybacteria) that diverged immediately following the origin of flagella. Proteasome evolution shows that the universal root is outside a clade comprising neomura and Actinomycetales (proteates), and thus lies within other eubacteria, contrary to a widespread assumption that it is between eubacteria and neomura. Cell wall and flagellar evolution independently locate the root outside Posibacteria (Actinobacteria and Endobacteria), and thus among negibacteria with two membranes. Posibacteria are derived from Eurybacteria and ancestral to neomura. RNA polymerase and other insertions strongly favour the monophyly of Gracilicutes (Proteobacteria, Planctobacteria, Sphingobacteria, Spirochaetes). Evolution of the negibacterial outer membrane places the root within Eobacteria (Hadobacteria and Chlorobacteria, both primitively without lipopolysaccharide): as all phyla possessing the outer membrane beta-barrel protein Omp85 are highly probably derived, the root lies between them and Chlorobacteria, the only negibacteria without Omp85, or possibly within Chlorobacteria. CONCLUSION: Chlorobacteria are probably the oldest and Archaebacteria the youngest bacteria, with Posibacteria of intermediate age, requiring radical reassessment of dominant views of bacterial evolution. The last ancestor of all life was a eubacterium with acyl-ester membrane lipids, large genome, murein peptidoglycan walls, and fully developed eubacterial molecular biology and cell division. It was a non-flagellate negibacterium with two membranes, probably a photosynthetic green non-sulphur bacterium with relatively primitive secretory machinery, not a heterotrophic posibacterium with one membrane.

Journal Article↗
PubMed1993

Gene deletion suggests a role for Trypanosoma cruzi surface glycoprotein GP72 in the insect and mammalian stages of the life cycle.

We have explored the biological function of a surface glycoprotein (GP72) of Trypanosoma cruzi by studying a null mutant parasite, generated by targeted gene deletion. GP72 deletion affected parasite morphology in several stages of the life cycle. Insect midgut (epimastigote) forms had a detached flagellum (apomastigote) in the null mutant. The abnormal flagellar phenotype persisted during development of the infective (metacyclic) forms but there was no impairment in the acquisition of complement resistance, sialidase expression or cell infectivity. The GP72 null mutant could efficiently infect and proliferate in mouse macrophages and non-phagocytic L6E9 cells. The mammalian stages of the life cycle also showed major morphological abnormalities. During early subcultures in L6E9 cells, few extracellular fully flagellated forms, expressing markers characteristic of trypomastigotes, were seen. The extracellular population consisted almost exclusively of rounded forms with short flagella (micromastigote), which expressed an amastigote-specific surface marker and no sialidase. The propagation of the parasite was not affected, despite the apparent lack of the trypomastigote forms, which are thought to be primarily responsible for cell invasion. After some subcultures, the extracellular population changed to about equal numbers of micromastigotes and a range of flagellated forms that still did not include true trypomastigotes. Instead, the kinetoplast remained close to the nucleus and the flagellum emerged from the middle of the cell (mesomastigote). Half of the flagellum adhered to the cell body and the remainder was free at the anterior end. In Triatoma infestans, the survival of the mutant was dramatically reduced, suggesting that either GP72 itself, or the altered properties of the flagellum, were critical for establishment in the insect vector.

Animals↗
PubMed1999

Cloning, sequencing, and nucleolar targeting of the basal-body-binding nucleolar protein BN46/51.

BN46/51 is an acidic protein found in the granular component of the nucleolus of the amebo-flagellate Naegleria gruberi. When Naegleria amebae differentiate into swimming flagellates, BN46/51 is found associated with the basal body complex at the base of the flagella. In order to determine the factors responsible for targeting BN46/51 to a specific subnucleolar region, cDNAs coding for both subunits were isolated and sequenced. Two clones, JG4.1 and JG12.1 representing the 46 kDa and 51 kDa subunits, respectively, were investigated in detail. JG12.1 encoded a polypeptide of 263 amino acids with a predicted size of 30.1 kDa that co-migrated with the 51 kDa subunit of BN46/51 when expressed in yeast. JG4.1 encoded a polypeptide of 249 amino acids with a predicted size of 28.8 kDa that co-migrated with the 46 kDa subunit of BN46/51. JG4.1 was identical to JG12.1 except for the addition of an aspartic acid between positions 94 and 95 of the JG12.1 sequence and the absence of 45 amino acids beginning at position 113. The predicted amino acid sequences were not closely related to any previously reported. However, the sequences did have 26-31% identity to a group of FKPBs (FK506 binding proteins) but lacked the peptidyl-prolyl cis-trans isomerase domain of the FKBPs. Both subunits contained two KKE and three KKX repeats found in other nucleolar proteins and in some microtubule binding proteins. Using 'Far Western' blots of nucleolar proteins, BN46/51 bound to polypeptides of 44 kDa and 74 kDa. The 44 kDa component was identified as the Naegleria homologue of fibrillarin. BN46/51 bound specifically to the nucleoli of fixed mammalian cells, cells which lack a BN46/51 related polypeptide. When the JG4.1 and JG12.1 cDNAs were expressed in yeast, each subunit was independently targeted to the yeast nucleolus. We conclude that BN46/51 represents a unique nucleolar protein that can form specific complexes with fibrillarin and other nucleolar proteins. We suggest that the association of BN46/51 with the MTOC of basal bodies may reflect its role in connecting the nucleolus with the MTOC activity for the mitotic spindle. This would provide a mechanism for nucleolar segregation during the closed mitosis of Naegleria amebae.

3T3 Cells↗
PubMed1975

An ultrastructural study of the spermatozoid of the fern, Marsilea vestita.

The ultrastructure of the mature spermatozoid of Marsilea vestita was studied after its release from the microspore and prior to its penetration of the egg. The psermatozoid is a pear-shaped cell with a complex spiral structure coiled around the edge in the narrow anterior end. This coil is composed of a large mitochondrion, elongated nucleus with highly condensed chromatin, a ribbon of microtubules, and a dense band of material (flagellated band) into which the flagella are inserted. There are over a hundred flagella protruding from each spermatozoid along the length of the coil. At the anterior tip of the coil is a short multilayered structure. It is not known what maintains the helical shape of the coil. The microtubular ribbon could be involved, but it is also possible that either the flagellated band, the condensed chromatin, or both, are sufficiently rigid to retain their shpaes unaided. When the spermatozoid is first released from the microspore it includes a cytoplasmic vesicle in the posterior region containing plastids, mitochondria, and other organelles. This vesicle is shed, taking the nuclear envelope with it, before the spermatozoid reaches the egg.

Cell Nucleus↗
PubMed1981

Studies of the rhizoplast from Naegleria gruberi.

A procedure utilizing homogenization and centrifugation in a low ionic strength buffer containing Triton X-100, has been used to facilitate the isolation of the rhizoplast from flagellates of Naegleria gruberi. This has enabled a study to be made of the physical and biochemical properties of this organelle. The rhizoplast is shown to be a proteinaceous structure with chemical properties similar to those of the molluscan gill ciliary rootlet. Polyacrylamide gel electrophoresis gives a possible subunit molecular weight of approximately 240 000 Daltons. Studies with antisera raised against the rhizoplast fraction demonstrated the absence of rhizoplast antigens in amoeboid forms of Naegleria gruberi and is taken as evidence that the organelle is synthesized de novo during transformation of the amoeba to the flagellate form. Results of optical diffraction studies on isolated rhizoplasts are also presented.

Amoeba↗
PubMed1981

Development of the cell wall in Tetraselmis: role of the Golgi apparatus and extracellular wall assembly.

The green algal flagellate, Tetraselmis, is a key transition organism in the phylogeny of green algae. It has been proposed that the cell wall of Tetraselmis arose evolutionarily from the fusion of scales and that this event secondarily caused the alteration of some cytoplasmic processes such as mitotic and cytokinetic mechanisms. Ultrastructural and developmental studies of the cell wall were performed with several strains of Tetraselmis. Two major wall types are reported. The wall of type 1 cells consists of a thick inner region covered by a layer of regularly repeating subunits of 26 nm, comparable to the subunits found in the median W2-W6 layer of Chlamydomonas. The more elaborate type 2 cell wall consists of a thick median wall layer, homologous to the type 1 inner wall, with additional inner and outer strata of hairs, grains and scales. Development of the cell wall begins in the endomembrane system, particularly the Golgi apparatus, where fibrillar tufts and electron-dense droplets are synthesized, modified and transported to the outside. Here, the tufts and droplets are displaced around the protoplast and assemble in several steps to yield the intact wall. Edge-growth assembly of the wall occurs here synchronously with cytoplasmic developments to yield the characteristic anterior flagellar pit. Models explaining various aspects of this development are discussed. When released from the cell, the wall subunits are not completely comparable to stellate scales, but appear to correspond to developmental stages of scales in green flagellates possessing body scales.

Biological Evolution↗
PubMed2006

Microbiological monitoring of laboratory mice and biocontainment in individually ventilated cages: a field study.

Over recent years, the use of individually ventilated cage (IVC) rack systems in laboratory rodent facilities has increased. Since every cage in an IVC rack may be assumed to be a separate microbiological unit, comprehensive microbiological monitoring of animals kept in IVCs has become a challenging task, which may be addressed by the appropriate use of sentinel mice. Traditionally, these sentinels have been exposed to soiled bedding but more recently, the concept of exposure to exhaust air has been considered. The work reported here was aimed firstly at testing the efficiency of a sentinel-based microbiological monitoring programme under field conditions in a quarantine unit and in a multi-user unit with frequent imports of mouse colonies from various sources. Secondly, it was aimed at determining biocontainment of naturally infected mice kept in an IVC rack, which included breeding of the mice. Sentinels were exposed both to soiled bedding and to exhaust air. The mice which were used in the study carried prevalent infectious agents encountered in research animal facilities including mouse hepatitis virus (MHV), mouse parvovirus (MPV), intestinal flagellates and pinworms. Our data indicate that the sentinel-based health monitoring programme allowed rapid detection of MHV, intestinal flagellates and pinworms investigated by a combination of soiled bedding and exhaust air exposure. MHV was also detected by exposure to exhaust air only. The IVC rack used in this study provided biocontainment when infected mice were kept together with non-infected mice in separate cages in the same IVC rack.

Animal Husbandry↗
PubMed1975

Changes in lipase and phosphatase activities of rat spermatozoa in transit from the caput to the cauda epididymidis.

Rat spermatozoa from the cauda epididymidis, freed from their cytoplasmic droplets and acrosomes, were found to have a lower lipid content and to incorporate [14C]glucose into their glycerides and glycerophosphatides at a lower rate than spermatozoa from the caput epididymidis. Against the background of the activities of some glycolytic enzymes which remained constant the activity of alkaline phosphatase decreased in spermatozoa migrating through the epididymis, whereas the activity of monoglyceride lipase increased. The corresponding enzyme activities of non-flagellate germ cells of the testis were measured for comparison. The triglyceride lipase of non-flagellate germ cells and of spermatozoa from both caput and cauda epididymidis was activated by cyclic 3':5'-AMP.

Acid Phosphatase↗
PubMed1992

[A trial designed to obtain a specific pathogen free Syrian hamster colony by administration of chemicals].

Conventional Syrian hamsters, contaminated with Giardia spp., Spironucleus muris, Trichomonas spp., Pasteurella pneumotropica and Pseudomonas aeruginosa were treated with chemicals in order to obtain specific pathogen free animals. Hamsters kept in the laminar flow rack were treated orally with metronidazole several times to obtain a flagellate-free colony. After all flagellates had been eradicated, one pair of animals were kept in an isolator and mating was allowed to occur. When their offspring reached the age of seven weeks, they were intramuscularly injected daily with netilmicin sulfate for 10 consecutive days. Following these treatments, all of the hamsters were free of Pasteurella and Pseudomonas. Further breeding of these animals was continued in isolators. To confirm the absence of selected pathogens, they were placed in a barrier room for further breeding as specific pathogen free animals.

Animals↗
PubMed2000

A novel express bioassay for detecting toxic substances in water by recording rhodopsin-mediated photoelectric responses in Chlamydomonas cell suspensions.

The influence of Cu2+, Zn2+, Cd2+, Pb2+ and formaldehyde on rhodopsin-mediated photoelectric responses in the green flagellate Chlamydomonas reinhardtii was investigated using three modifications of a recently developed population method for electrical recording (in nonoriented, phototactically preoriented (PO) and gravitactically preoriented cell suspensions). The addition of the heavy metal ions at concentrations several times lower than those known to affect swimming velocity and other physiological parameters in photosynthetic flagellates led to a rapid (one to several minutes) inhibition of the responses. Formaldehyde induced a significant temporary increase in the gravi-orientation of the cells simultaneously with an inhibition of their photoelectric cascade, photo-orientation and motility. The signals recorded in PO suspensions were more sensitive to all tested toxic substances than those recorded from nonoriented cells and indicated a switch from negative to positive phototaxis in the presence of the toxic substances. Of the two major components of the photoelectric cascade, the regenerative response was more sensitive to the tested heavy metal ions, but not to formaldehyde, than the photoreceptor current. The results obtained show that measurement of the photoinduced electrical responses in Chlamydomonas cell suspensions is a powerful novel bioassay for testing environmental pollutants in water samples.

Animals↗
PubMedDate not supplied

The whole is more than the sum of its parts: Modeling community-level effects of UVR in marine ecosystems.

The effect of UVB radiation (UVBR, 290-320 nm) on the dynamics of the lower levels of the marine plankton community was modeled. The model was built using differential equations and shows a good fit to experimental data collected in mesocosms (defined as large enclosures of 1500 L filled with natural marine waters). Some unexpected results appear to be possible by indirect effects in prey (bacteria, phytoplankton and heterotrophic flagellates). In particular, apparent competition appears between small phytoplankton and bacteria. This effect is caused by a shared predator (ciliates). Another remarkable effect is an increase in bacteria and flagellates populations due to enhanced UVBR. This effect is similar to that observed under mesocosm experimental conditions and is related to the decrease of predation due to the direct damage to predators (ciliates) by UVBR. The effect of UVBR changing interaction coefficients may be dramatic on the community structure, producing big changes in equilibrium populations, as demonstrated by sensitivity analysis of the model. In order to generalize these results to field conditions it will be necessary to increase model complexity and include extra organic mater sources, mixing and sinking effects and predation by large zooplankton. This work shows that UVBR may produce community global responses that are consequence of both direct and indirect effects among populations.

Ecosystem↗
PubMedDate not supplied

Morphological evidence by scanning electron microscopy of excretion of metacyclic forms of Trypanosoma cruzi in vector's urine.

Comparison by scanning electron microscopy (SEM) of Trypanosoma cruzi flagellates attached to the cuticle of the rectal gland of infected Dipetalogaster maxima nymphs, showed marked differences before and after feeding. Before feeding numerous metacyclic trypomastigotes were observed among the abundant epimastigotes that formed the carpet of flagellates. On the other hand, in insects that were allowed to urinate for 24 hours after a meal, the metacyclics were scarce, indicating that they had been detached by the urine flow. An asymmetric type of cell division, probably originating both an epi- and a trypomastigote, was occasionally observed. The occurrence of swellings at different levels of the flagella of epimastigotes suggests that secondary sites of attachment may be common.

Animals↗
PubMedDate not supplied

Parasite genotypically related to a monoxenous trypanosomatid of dog's flea causing opportunistic infection in an HIV positive patient.

An HIV positive patient presenting a clinical picture of visceral leishmaniasis co-infection was submitted to a bone marrow aspiration after admission to hospital. Amastigotes forms were seen in the bone marrow aspirate and the parasite grew in culture as promastigotes. Molecular analyses showed that the flagellates isolated did not belong to the genera Leishmania, Trypanosoma or Sauroleishmania. It was not possible to establish infection in laboratory animals. In vitro culture of mouse peritoneal macrophages revealed the invasion of the host cells by the flagellates and their killing 48 hr after infection. Opportunistic infection with an insect trypanosomatid was suspected. Further hybridization analyses against a panel of different monoxenous and heteroxenous trypanosomatids showed kDNA cross-homology with Leptomonas pulexsimulantis a trypanosomatid found in the dog's flea.

AIDS-Related Opportunistic Infections↗
PubMed2005

Peridomiciliary colonies of Triatoma vitticeps (Stal, 1859) (Hemiptera, Reduviidae, Triatominae) infected with Trypanosoma cruzi in rural areas of the state of Espírito Santo, Brazil.

In Brazil, the colonization of human dwellings by triatomines occurs in areas with native vegetation of the caatinga or cerrado types. In areas of Atlantic forest such as in the Brazilian state of Espírito Santo, there are no species adapted to live in human habitations. The few autochthonous cases of Chagas disease encountered in Espírito Santo have been attributed to adult specimens of Triatoma vitticeps that invade houses from forest remnants. In recent years, the entomology unit of the Espírito Santo State Health Secretariat has recorded nymphs infected with flagellates similar to Trypanosoma cruzi in rural localities. Entomological surveys were carried out in the residences and outbuildings in which the insects were found, and serological examinations for Chagas disease performed on the inhabitants. Four colonies were found, all associated with nests of opossums (Didelphis aurita), 111 specimens of T. vitticeps, and 159 eggs being collected. All the triatomines presented flagellates in their frass. Mice inoculated with the faeces presented trypomastigotes in the circulating blood and groups of amastigotes in the cardiac muscle fibres. Serological tests performed on the inhabitants were negative for T. cruzi. Even with the intense devastation of the forest in Espírito Santo, there are no indications of change in the sylvatic habits of T. vitticeps. Colonies of this insect associated with opossum nests would indicate an expansion of the sylvatic environment into the peridomicile.

Animals↗
PubMed2007

[Standardization of conditions for PCR detection of Leishmania spp. DNA in sand flies (Diptera, Psychodidae)].

The correct identification of etiological agents in vector insects is crucial for epidemiological studies. Identification of flagellates in such vectors, usually by dissection of the digestive tract and microscopic observation of the contents as well as attempts at parasite isolation from insects in culture media, have proven operationally inadequate and with poor diagnostic specificity, since female sand flies are also hosts for other flagellates like Trypanosoma and Endotrypanum. Due to the efficiency and specificity of DNA target sequence amplification by polymerase chain reaction (PCR), the latter could be used to investigate the presence of Leishmania in sand flies, although the insects need to be properly stored and the Leishmania DNA extracted using appropriate methodology. This paper describes methodologies to standardize sand fly storage and Leishmania DNA extraction in such specimens as a more practical method in field studies.

Animals↗
PubMed2003

Tritrichomonas foetus and not Pentatrichomonas hominis is the etiologic agent of feline trichomonal diarrhea.

Recently, several investigators have reported large-bowel diarrhea in cats associated with intestinal trichomonad parasites. These reports have presumptively identified the flagellates as Pentatrichomonas hominis, a n organism putatively capable of infecting the intestinal tracts of a number of mammalian hosts, including cats, dogs, and man. The purpose of the present study was to determine the identity of this recently recognized flagellate by means of rRNA gene sequence analysis; restriction enzyme digest mapping; and light, transmission, and scanning electron microscopy (SEM).

Animals↗