Validation of putative biomarkers of furan exposure through quantitative analysis of furan metabolites in urine of F344 rats exposed to stable isotope labeled furan.

Humans are chronically exposed to furan, a potent liver toxicant and carcinogen that occurs in a variety of heat-processed foods. Assessment of human exposure based on the furan content in foods is, however, subject to some uncertainty due to the high volatility of furan. Biomarker monitoring is thus considered an alternative or complementary approach to furan exposure assessment. Previous work suggested that urinary furan metabolites derived from the reaction of cis-2-butene-1,4-dial (BDA), the reactive intermediate of furan, with glutathione (GSH) or amino acids may serve as potential biomarkers of furan exposure. However, some metabolites were also reported to occur in urine of untreated animals, indicating either background contamination via animal feed or endogenous sources, which may limit their suitability as biomarkers of exposure. The overall aim of the present study was to accurately establish the correlation between external dose and concentration of furan metabolites in urine over time and to discriminate against endogenous formation and furan intake via feed. To this end, the furan metabolites GSH-BDA (N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine), NAcLys-BDA (R-2-(acetylamino)-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid), NAcCys-BDA-NAcLys (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine) and NAcCys-BDA-NAcLys sulfoxide (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine sulfoxide) were simultaneously analyzed by stable isotope dilution ESI-LC-MS/MS as unlabeled and [13C4]-furan dependent metabolites following oral administration of a single oral dose of isotopically labelled [13C4]-furan (0.1, 1, 10, 100 and 1000 µg/kg bw) to male and female F344/DuCrl rats. Although a linear correlation between urinary excretion of [13C4]-furan-dependent metabolites was observed, analysis of unlabeled NAcLys-BDA, NAcCys-BDA-NAcLys and NAcCys-BDA-NAcLys sulfoxide revealed substantial, fairly constant urinary background levels throughout the course of the study. Analysis of furan in animal feed excluded feed as a source for these background levels. GSH-BDA was identified as the only furan metabolite without background occurrence, suggesting that it may present a specific biomarker to monitor external furan exposure. Studies in humans are now needed to establish if analysis of urinary GSH-BDA may provide reliable exposure estimates.

Dynamic ventral disc contraction is necessary for Giardia attachment and host pathology.

Giardia lamblia is a common parasitic protist that infects the small intestine and causes giardiasis, resulting in diarrhea, vomiting, weight loss, and malabsorption. Giardiasis leads to cellular damage, including loss of microvilli, disruption of tight junctions, impaired barrier function, enzyme inhibition, malabsorption, and apoptosis. In the host, motile Giardia trophozoites attach to the duodenal microvilli using a unique microtubule organelle called the ventral disc. Despite early observations of disc-shaped depressions in microvilli after parasite detachment, little is known about disc-mediated attachment mechanisms and there little direct evidence showing that parasite attachment causes cellular damage. However, advancements in in vitro organoid models of infection and genetic tools have opened new possibilities for studying molecular mechanisms of attachment and the impact of attachment on the host. Through high-resolution live imaging and a novel disc mutant, we provide direct evidence for disc contraction during attachment, resolving the long-standing controversy of its existence. Specifically, we identify three types of disc movements that characterize contraction, which in combination result in a decrease in disc diameter and volume. Additionally, we investigate the consequences of attachment and disc contractility using an attachment mutant that has abnormal disc architecture. In a human organoid model, we demonstrate that this mutant has a limited ability to break down the epithelial barrier as compared to wild type. Based on this direct evidence, we propose a model of attachment that incorporates disc contraction to generates the forces required for the observed "grasping" of trophozoites on the host epithelium. Overall, this work highlights the importance of disc contractility in establishing and maintaining parasite attachment, leading to intestinal barrier breakdown.

Can high resolution micro-ultrasound replace MRI in the diagnosis of prostate cancer?

High resolution micro-ultrasound (micro-u/s) is a novel technology that permits visualization of lesions suspicious for prostate cancer. The resolution of 70 µ, that of a prostatic duct, means that alterations in ductal anatomy and cellular density are readily apparent. Initial experience in multiple centers comparing it to mpMRI suggests that the sensitivity for clinically significant prostate cancer is comparable or superior. Specificity is comparable or mildly reduced. Micro-u/s is an inexpensive, accessible and convenient alternative to mpMRI for imaging and diagnosing prostate cancer.

Giardia duodenalis assemblages in Egyptian children with diarrhea.

Giardia duodenalis is considered the most common cause of parasitic diarrhea worldwide. Genetic studies revealed that at least eight assemblages (A-H) exist. Of these assemblages, A and B are found primarily in human beings and occasionally in animals. The association between clinical symptoms and G. duodenalis assemblages is controversial. The aim of the present study was to determine the assemblages of G. duodenalis prevalent among Egyptian children with diarrhea. Therefore, 96 positive stool samples for Giardia by light microscopy were subjected to multilocus genotyping targeting the triose phosphate isomerase (tpi), ß-giardin (bg), and glutamate dehydrogenase (gdh) genes. Amplified polymerase chain reaction (PCR) products were then purified, sequenced, and aligned with reference strains to determine the assemblages of the Giardia isolates. Out of the 96 microscopically positive stool samples for Giardia, 77 (80 %) were successfully amplified and sequenced at least at one locus. Of these, 21 (27.3 %) were shown to be assemblage A, 54 (70.1 %) assemblage B, while discordant sequence typing results were observed in 2 (2.6 %) samples. AII was the predominant subassemblage of assemblage A, while it was generally difficult to further classify assemblage B. It was concluded that infection with assemblage B was more common than that with assemblage A. No associations between epidemiological information and assemblage were detected, except with age. Although infections with assemblage B were more frequently associated with abdominal pain and acute diarrhea than with assemblage A, the difference was not statistically significant.

Detection of Giardia duodenalis assemblage A and B isolates by immunochromatography in stool samples from Rwandan children.

We evaluated the performance of an immunochromatographic assay (ICA) in comparison with light microscopy and PCR for the detection of Giardia duodenalis in stool samples from 558 Rwandan children. The association of infection with clinical symptoms was similar for the three diagnostic tools. The ICA equally detected parasites of assemblages A and B and was more sensitive than light microscopy (50.4 versus 29.5% of PCR-positive samples considered true positive; p <0.0001). Hence, the ICA shows superior sensitivity compared with microscopy but still misses half of the G. duodenalis infections detected by PCR in this hyperendemic area.

[Fever as the presenting manifestation of colon cancer: a case series of 11 patients]. / Fièvre révélatrice d'un cancer colique: 11 observations.

INTRODUCTION: Fever happens frequently in colon cancer but it is rarely the presenting manifestation. PATIENTS AND METHODS: We report a case series of patients with colon cancer revealed by fever in the three military hospitals in Paris. RESULTS: Of the 11 patients studied, seven were men and four were women, and their mean age was 70 years. Cancer was localized in the sigmoid colon (n=6), left colon (n=3) and right colon (n=2). Cancer staging (UICC TNM classification 2002) was respectively pTis (n=1), I (n=4), II (n=3) and III (n=3). Fever was the only reason for admission and two patients had a recurrent fever of unknown origin. All patients but one had bacterial infection. Blood cultures grew up in six cases, Escherichia coli (n=3), Streptococcus gallolyticus (ex bovis) (n=2) and anaerobic bacteria (n=1). There was one case of infective endocarditis caused by S. gallolyticus. Imaging showed a liver abscess (n=3) and a colon cancer complicated by an abscess (n=3). In seven patients, a familial history of colon cancer and symptoms of underlying colic disease were present (neglected rectal bleeding, iron deficiency anaemia, clinical evidence of an abdominal mass). CONCLUSIONS: Fever may reveal colon cancer at an early stage. Its main cause is a bacterial infection, such as bacteremia or abscess. Fever of unknown origin is a rare presentation. Detailed history, careful clinical examination and analysis of imaging contribute to recommend the prescription of colonoscopy.

Bug22p, a conserved centrosomal/ciliary protein also present in higher plants, is required for an effective ciliary stroke in Paramecium.

Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate.

Genetic evidence for interaction between eta- and beta-tubulins.

The thermosensitive allelic mutations sm19-1 and sm19-2 of Paramecium tetraurelia cause defective basal body duplication: growth at the nonpermissive temperature yields smaller and smaller cells with fewer and fewer basal bodies. Complementation cloning of the SM19 gene identified a new tubulin, eta-tubulin, showing low homology with each of the other five tubulins, alpha to epsilon, characterized in P. tetraurelia. In order to analyze eta-tubulin functions, we used a genetic approach to identify interacting molecules. Among a series of extragenic suppressors of the sm19-1 mutation, the su3-1 mutation was characterized as an E288K substitution in the beta-PT2 gene coding for a beta-tubulin, while the mutation nocr1 conferring nocodazole resistance and localized in another beta-tubulin gene, beta-PT3, was shown to enhance the mutant phenotype. The interaction between eta-tubulin and microtubules, revealed by genetic data, is supported by two further types of evidence: first, the mutant phenotype is rescued by taxol, which stabilizes microtubules; second, molecular modeling suggests that eta-tubulin, like gamma- and delta-tubulins, might be a microtubule minus-end capping molecule. The likely function of eta-tubulin as part of a complex specifically involved in basal body biogenesis is discussed.

KIN241: a gene involved in cell morphogenesis in Paramecium tetraurelia reveals a novel protein family of cyclophilin-RNA interacting proteins (CRIPs) conserved from fission yeast to man.

In this study, we report cloning, by functional complementation of the KIN241 gene involved in Paramecium cell morphogenesis, cortical organization and nuclear reorganization. This gene is predicted to encode a protein of a novel type, comprising a cyclophilin-type, peptidyl-prolyl isomerase domain, an RNA recognition motif, followed by a region rich in glutamate and lysine (EK domain) and a C-terminal string of serines. As homologues of this protein are present in the genomes of Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana and Homo sapiens, the Kin241p predicted sequence defines a new family of proteins that we propose to call 'CRIP', for cyclophilin-RNA interacting protein. We demonstrate that, in Paramecium, Kin241p is localized in the nucleus and that deletion of some nuclear localization signals (NLSs) decreases transport of the protein into the nucleus. No Kin241-1 protein is present in mutant cells, suggesting that the C-terminal serine-rich region is responsible for protein stability.

Role of delta-tubulin and the C-tubule in assembly of Paramecium basal bodies.

BACKGROUND: A breakthrough in the understanding of centriole assembly was provided by the characterization of the UNI3 gene in Chlamydomonas. Deletion of this gene, found to encode a novel member of the tubulin superfamily, delta-tubulin, results in the loss of the C-tubule, in the nine microtubule triplets which are the hallmark of centrioles and basal bodies. Delta-tubulin homologs have been identified in the genomes of mammals and protozoa, but their phylogenetic relationships are unclear and their function is not yet known. RESULTS: Using the method of gene-specific silencing, we have inactivated the Paramecium delta-tubulin gene, which was recently identified. This inactivation leads to loss of the C-tubule in all basal bodies, without any effect on ciliogenesis. This deficiency does not directly affect basal body duplication, but perturbs the cortical cytoskeleton, progressively leading to mislocalization and loss of basal bodies and to altered cell size and shape. Furthermore, additional loss of B- and even A-tubules at one or more triplet sites are observed: around these incomplete cylinders, the remaining doublets are nevertheless positioned according to the native ninefold symmetry. CONCLUSIONS: The fact that in two distinct phyla, delta-tubulin plays a similar role provides a new basis for interpreting phylogenetic relationships among delta-tubulins. The role of delta-tubulin in C-tubule assembly reveals that tubulins contribute subtle specificities at microtubule nucleation sites. Our observations also demonstrate the existence of a prepattern for the ninefold symmetry of the organelle which is maintained even if less than 9 triplets develop.

Basal body-associated nucleation center for the centrin-based cortical cytoskeletal network in Paramecium.

The infraciliary lattice, a contractile cortical cytoskeletal network of Paramecium, is composed of a small number of polypeptides including centrins. Its overall pattern reflects a hierarchy of structural complexity, from assembly and bundling of microfilaments to formation of polygonal meshes arranged in a continuous network subtending the whole cell surface, with local differentiations in the shape and size of the meshes. To analyse how the geometry of this complex network is generated and maintained, we have taken two approaches. Firstly, using monoclonal antibodies raised against the purified network, we have shown that all the component polypeptides colocalize, in agreement with previous biochemical data indicating that the infraciliary lattice is formed of large complexes comprising all the component polypeptides. Secondly, by taking advantage of different experimental conditions leading to disassembly of the network, we have followed its reassembly. Cytological analysis of the process revealed 1) that the network regrows exclusively from specific infraciliary lattice organizing centers (ICLOC), precisely localized near each basal body and, 2) that the global organization is not precisely controlled by genetic information but by the basal body pattern. Finally, slight ultrastuctural differences between reassembled and control lattices suggest that the organization of the filament bundles is partly templated by that of the preexisting ones.

The SM19 gene, required for duplication of basal bodies in Paramecium, encodes a novel tubulin, eta-tubulin.

The discovery of delta-tubulin, the fourth member of the tubulin superfamily, in Chlamydomonas [1] has led to the identification in the genomes of vertebrates and protozoa of putative delta homologues and of additional tubulins, epsilon and zeta [2-4]. These discoveries raise questions concerning the functions of these novel tubulins, their interactions with microtubule arrays and microtubule-organising centres, and their evolutionary status. The sm19-1 mutation of Paramecium specifically inhibits basal body duplication [5] and causes delocalisation of gamma-tubulin, which is also required for basal body duplication [6]. We have cloned the SM19 gene by functional complementation and found that it encodes another new member of the tubulin superfamily. SM19p, provisionally called eta-tubulin (eta-tubulin), shows low sequence identity with the tubulins previously identified in Paramecium, namely, alpha [7], beta [8], gamma [6], delta (this work) and epsilon (P. Dupuis-Williams, personal communication). Phylogenetic analysis indicated that SM19p is not consistently grouped with any phylogenetic entity.

Homology-dependent gene silencing in Paramecium.

Microinjection at high copy number of plasmids containing only the coding region of a gene into the Paramecium somatic macronucleus led to a marked reduction in the expression of the corresponding endogenous gene(s). The silencing effect, which is stably maintained throughout vegetative growth, has been observed for all Paramecium genes examined so far: a single-copy gene (ND7), as well as members of multigene families (centrin genes and trichocyst matrix protein genes) in which all closely related paralogous genes appeared to be affected. This phenomenon may be related to posttranscriptional gene silencing in transgenic plants and quelling in Neurospora and allows the efficient creation of specific mutant phenotypes thus providing a potentially powerful tool to study gene function in Paramecium. For the two multigene families that encode proteins that coassemble to build up complex subcellular structures the analysis presented herein provides the first experimental evidence that the members of these gene families are not functionally redundant.

Genetic evidence for a role of centrin-associated proteins in the organization and dynamics of the infraciliary lattice in Paramecium.

Within the superfamily of "EF-hand Ca2+-modulated proteins," centrins constitute a family of cytoskeletal proteins that are highly conserved from lower eukaryotes to man. Their cytoskeletal specialization is manifest in their capacity to form filamentous contractile arrays of various shapes and functions and by their association with microtubule organizing centres (MTOCs). While the latter property has been conserved throughout the evolution of eukaryotes, centrin-based contractile structures are only found in protists where they form arrays of widely diverse organization and function. In the ciliate Paramecium tetraurelia, three centrin genes have been characterized, which may be part of a larger centrin gene family [Madeddu et al., 1996: Eur J. Biochem. 238:121-128]. The products of these genes were originally identified as components of the infraciliary lattice, a contractile cytoskeletal network [Garreau de Loubresse et al., 1991: Biol. Cell 71:217-225]. We show here that centrins are localized not only in this lattice but also in basal bodies and in the cord, a filamentous structure associated with the oral apparatus. We demonstrate that in the infraciliary lattice, but not in basal bodies, centrins are associated with high-molecular-weight proteins (ca. 350 kD). Their role in the biogenesis of the infraciliary lattice is documented by cytological and biochemical properties of the mutant "démaillé" (dem1) characterized by altered centrin-associated proteins and abnormal organization and dynamics of the infraciliary lattice.

Gamma-tubulin is permanently associated with basal bodies in ciliates.

Ciliates are of special interest owing to the multiplicity and diversity of their microtubule organizing centers (MTOCs). The subcellular localization of gamma-tubulin in these protozoa has not been extensively studied. The cloning of a gamma-tubulin gene in Euplotes (Liang, A., K. Heckmann, Gene 136, 319-322 (1993) led us to examine the localization of this protein. We used three polyclonal antibodies, JH46, R58 and R70. They had been raised against peptides common to mammalian and Aspergillus gamma-tubulins. These regions had 69%, 95%, and 75% identity with the corresponding regions of Euplotes gamma-tubulin. Immunoblotting (R70) revealed a polypeptide corresponding to the molecular mass of Euplotes gamma-tubulin. In Euplotes octocarinatus, gamma-tubulin was detected by immunofluorescence (R70) in the basal bodies, the micronucleus and the macronucleus throughout the cell cycle. The presence of gamma-tubulin in basal bodies and micronuclei was confirmed with the other two antibodies JH46 and R58. The permanent association of gamma-tubulin with basal bodies was also observed in Tetrahymena thermophila and Paramecium tetraurelia, two ciliates distantly related to Euplotes. These results not only extend to ciliates the finding that gamma-tubulin is permanently associated with ciliary basal bodies, but also demonstrate that gamma-tubulin is present in unconventional MTOCs.

Characterization of centrin genes in Paramecium.

Centrins are highly conserved, ubiquitous cytoskeletal components which belong to the EF-hand superfamily of Ca2+-modulated proteins. We report here the molecular characterization of new members of the centrin family, Paramecium centrins. Previous studies described the organization of the infraciliary lattice (ICL), the innermost cortical cytoskeletal network of Paramecium, and showed that it was composed of a set of low-molecular-mass, Ca2+-binding polypeptides [Garreau de Loubresse, N., Klotz, C., Vigues, B., Rutin, J & Beisson, J. (1991) Biol. Cell 71, 217-225]. In this paper we show that these polypeptides are recognized by specific anti-centrin polyclonal antibodies. Their microsequences revealed four distinct N-termini. For one of them, ICL1, N-terminal and internal peptide sequences were used for PCR amplification and cloning of a DNA fragment containing a complete centrin coding sequence. The deduced amino acid sequence presents about 50% identify with those of centrins from other species. Further molecular analysis allowed us to identify two additional closely related, co-expressed ICL1 genes, providing the first example of a centrin multigenic family in a microorganism.

The tubulin gene family of Paramecium: characterization and expression of the alpha PT1 and alpha PT2 genes which code for alpha-tubulins with unusual C-terminal amino acids, GLY and ALA.

The ciliated protozoan Paramecium harbours a particularly large diversity of microtubule networks, ranging from the elaborate and stable ciliary axonemes and basal bodies to very dynamic cytoplasmic, cortical or intranuclear arrays. Their organization and individual cycle of assembly/disassembly are well known and extensive immunocytochemical studies of the post-translational modifications in the various microtubule systems have been reported. However, in order to better understand the biogenesis of these multiple and diverse microtubule arrays, it seemed necessary to characterize the tubulin gene family. We show that P tetraurelia possesses four alpha- and three beta-genes and we report the cloning and sequencing of two intronless alpha-genes, alpha PT1 and alpha PT2, which code for very similar polypeptides, differing only by their unusual C-terminal amino acids, respectively GLY and ALA. Partial sequencing of the two other alpha-genes suggests an absence of any further isotype diversity. In an attempt to study the expression of alpha PT1 and alpha PT2, polyclonal antibodies were raised against the twelve C-terminal amino acids corresponding to the deduced polypeptide sequences. The reactivity of these anti-sequence antibodies was studied on blots of soluble tubulin and in situ and compared with that of other well characterized anti-alpha-tubulin antibodies. The molecular data show that in Paramecium, like in other ciliates, microtubule diversity does not arise from tubulin isotype diversity. The immunocytological data indicate that the native C-terminal sequences are predominantly detected in transient or nascent microtubule arrays and lead us to propose: 1) that the C-terminal TYR, absent in Paramecium and in most cilate species, has no intrinsic functional role; and 2) that post-translational modifications do not seem directly instrumental in the geometry and functions of microtubule arrays.