Absence of genotoxic impact assessed by micronucleus frequency in circulating lymphocytes of workers exposed to cadmium.

PURPOSE: Cd is considered as a genotoxic carcinogen for which a threshold can be identified. This threshold has, however, not been established and the shape of the relationship between Cd exposure and genotoxic effects is unknown. The aim of the present study was to analyse the shape of the dose-response relationship for the genotoxic effects of Cd in occupational settings. METHODS: The study has a cross-sectional design and includes 60 healthy male and female workers with known Cd exposure selected from two plants manufacturing or recycling nickel-Cd batteries. The frequency of MN was measured in circulating lymphocytes, and related to internal Cd doses (Cd-B, Cd-U). Determinants of MN frequency were traced by multivariate regression analysis. RESULTS: Cd exposure covered a wide range as measured by Cd-B (0.02-1.26 µg/dL), Cd-U (0.26-15.80 µg/g creat) and seniority in the plant (1-42 years). Gender was the only parameter significantly associated with MN frequency, women having on average 8.5 additional MN/1000 BN cells compared to men. Cd-B, Cd-U or Ni-U did not influence MN frequency when adjusted for gender and other potential confounders. CONCLUSION: This finding is consistent with the existing knowledge on the mechanisms governing the genotoxic activity of Cd, which are all non-stochastic and thresholded. The threshold for systemic genotoxic effects of Cd is thus beyond the range of internal exposure considered in the present investigation.

Genetic interference in protozoa.

RNA interference first described in Caenorhabditis elegans and transgene-induced post-transcriptional gene silencing first described in plants and fungi now appear as different means of activating a conserved and ancient mechanism that can protect genomes against viruses and transposons and perhaps also control expression of endogenous genes. We present here similar genetic interference phenomena in highly divergent protozoa, Trypanosoma and Paramecium, and look ahead to what contribution these microorganisms could bring to this fast-moving area.

Flagellum ontogeny in trypanosomes studied via an inherited and regulated RNA interference system.

The African trypanosome, Trypanosoma brucei possesses a large and unique intraflagellar structure called the paraflagellar rod (PFR). The PFR is composed of 2 major proteins, PFRA and PFRC. We have generated an inducible mutant trypanosome cell line (snl-2) that expresses linked inverted copies of a PFRA gene, capable of forming a PFRA double-stranded (ds) RNA. When expression of this dsRNA was induced, new PFRA RNA and PFRA protein quickly disappeared and PFR construction was affected, resulting in cell paralysis. This inducible RNA interference (RNAi) effect was fast-acting, heritable and reversible. It allowed us to demonstrate that PFR proteins are able to enter both mature and growing flagella but appear to concentrate differentially in new flagella because of the construction process. The PFR is constructed by a polar assembly process at the distal end of the flagellum resulting in a stable cytoskeletal structure with low turn-over. The inducible RNAi approach will have widespread applicability in studies of gene function and cellular processes in parasites.

Inside and outside of the trypanosome flagellum:a multifunctional organelle.

Amongst the earliest eukaryotes, trypanosomes have developed conventional organelles but sometimes with extreme features rarely seen in other organisms. This is the case of the flagellum, containing conventional and unique structures whose role in infectivity is still enigmatic.

Flagellar morphogenesis: protein targeting and assembly in the paraflagellar rod of trypanosomes.

The paraflagellar rod (PFR) of the African trypanosome Trypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.

Protein transport and flagellum assembly dynamics revealed by analysis of the paralysed trypanosome mutant snl-1.

The paraflagellar rod (PFR) of Trypanosoma brucei is a large, complex, intraflagellar structure that represents an excellent system in which to study flagellum assembly. Molecular ablation of one of its major constituents, the PFRA protein, in the snl-1 mutant causes considerable alteration of the PFR structure, leading to cell paralysis. Mutant trypanosomes sedimented to the bottom of the flask rather than staying in suspension but divided at a rate close to that of wild-type cells. This phenotype was complemented by transformation of snl-1 with a plasmid overexpressing an epitope-tagged copy of the PFRA gene. In the snl-1 mutant, other PFR proteins such as the second major constituent, PFRC, accumulated at the distal tip of the growing flagellum, forming a large dilation or 'blob'. This was not assembled as filaments and was removed by detergent-extraction. Axonemal growth and structure was unmodified in the snl-1 mutant and the blob was present only at the tip of the new flagellum. Strikingly, the blob of unassembled material was shifted towards the base of the flagellum after cell division and was not detectable when the daughter cell started to produce a new flagellum in the next cell cycle. The dynamics of blob formation and regression are likely indicators of anterograde and retrograde transport systems operating in the flagellum. In this respect, the accumulation of unassembled PFR precursors in the flagellum shows interesting similarities with axonemal mutants in other systems, illustrating transport of components of a flagellar structure during both flagellum assembly and maintenance. Observation of PFR components indicate that these are likely to be regulated and modulated throughout the cell cycle.

A motility function for the paraflagellar rod of Leishmania parasites revealed by PFR-2 gene knockouts.

We demonstrate a functional role for the paraflagellar rod (PFR) in motility of Leishmania mexicana. The PFR is a complex cytoskeletal structure running parallel to the axoneme in the flagella of kinetoplastid protozoa. The PFR is composed of a latticework of protein filaments whose major constituents are two related proteins (PFR-1 and PFR-2 in Leishmania). The molecular details of their assembly into PFR filaments are unknown as is the biological function of the PFR. As an approach to understanding the structure and function of the PFR in Leishmania, we made L. mexicana null mutants of PFR-2. PFR-2 minus parasites grow and divide normally in culture and still express the PFR-1 protein. They lack most of the PFR structure demonstrating that the PFR-2 protein is an essential constituent of the PFR. Detailed ultrastructural analysis of the PFR-2 null mutant reveals the presence of a residual inner substructure of the PFR which contains PFR-1 protein, indicating that PFR-1 can polymerize in the absence of PFR-2. The PFR-2 null mutant displays pronounced changes in flagellar beat waveform and forward swimming velocity, compared to wild type parasites consistent with decreased internal elastic bending resistance in PFR-lacking flagella, and indicating a functional role for the PFR in the motility of Leishmania.

The paraflagellar rod of kinetoplastida: solved and unsolved questions.

The flagellum of almost every member of the Kinetoplastida contains, next to its canonical 'nine-plus-two' axoneme, structure, a unique, complex and highly organized lattice-like structure called the paraflagellar rod or paraxial rod. Here, Philippe Bastin, Keith Matthews and Keith Gull summarize the latest findings on its structure, the nature of its protein components and their corresponding genes. They also consider the possible functions of this intriguing organelle.

An M(r) 145,000 low-density lipoprotein (LDL)-binding protein is conserved throughout the Kinetoplastida order.

In view of the importance of the low-density lipoprotein (LDL)-receptor in Trypanosoma brucei, we have examined whether other bloodstream trypanosomes of medical and veterinary importance (T.b. rhodesiense, T. equiperdum, T. vivax, T. congolense), but also related parasites developing in mammalian (Leishmania donovani) and non-mammalian hosts (Crithidia luciliae and Phytomonas sp. isolated from Euphorbia), would possess an LDL-receptor of their own. (1) All these parasites specifically accumulate human 125I-LDL with a relatively 2.5-fold higher rate for bloodstream trypanosomes. (2) A mixture of monoclonal antibodies raised against T.b. brucei LDL-receptor inhibit binding of LDL to all species but with different efficiency. (3) A single glycoprotein of similar M(r) (gp145) is isolated by LDL-affinity chromatography from all the above species, as well as from both human serum-resistant and sensitive strain of T.b. rhodesiense, and from the bodonid member of the Kinetoplastida Trypanoplasma borelli. (4) Several control experiments including 35S-metabolic labeling of procyclic T.b. brucei and of C. luciliae followed by LDL-affinity chromatography or immunoprecipitation demonstrate that gp145 is indeed synthesised by the parasites and is not a contaminant of the experimental system. (5) In immunoblots and ELISA, these gp145 cross-react with the polyclonal and monoclonal antibodies raised against the LDL-receptor of T.b. brucei, the highest degree of cross-reactivity being found among the members of the Trypanozoon subgroup. (6) Finally, immunisation of mice with the purified LDL-receptor from one strain of T.b. brucei is not sufficient to confer durable protection against another strain of this parasite.

Activity, pharmacological inhibition and biological regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Trypanosoma brucei.

Activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, the key enzyme in the biosynthesis of steroids and polyisoprenoids in mammalian cells, has been detected in both the bloodstream form and the culture-adapted procyclic form of Trypanosoma brucei (3.7 +/- 0.6 and 12.7 +/- 1.8 pmol mevalonate produced min-1 (mg cell protein)-1, respectively). The enzyme activity is enriched 6-fold in microsomal fractions. Several competitive inhibitors of mammalian HMG-CoA reductase, including synvinolin (simvastatin), inhibit the multiplication of both forms of trypanosome in vitro (IC50, approx. 25-50 microM after 2-3 days). This growth inhibition is potentiated by agents interfering with the exogenous supply of cholesterol, such as antibodies blocking the low-density lipoprotein (LDL) receptor, or 5 microM chloroquine. Conversely, growth inhibition by synvinolin can be largely reverted either by 300 nM LDL or by products of the mevalonate pathway, such as 20 mM mevalonate and in procyclics by 100 microM squalene or cholesterol. In procyclics, low concentrations of synvinolin selectively inhibit the incorporation of [14C]acetate into sterols, but not into fatty acids. These results argue for a critical role in trypanosomes of a mevalonate pathway, that is involved in the biosynthesis of sterol and probably of other metabolites. The HMG-CoA reductase activity is decreased 2-fold in procyclics incubated with 4 mM mevalonate and increased 2-fold in the presence of 2.5 microM synvinolin. Synvinolin also upregulates LDL binding up to 4-fold. These data suggest that HMG-CoA reductase and LDL receptor expression are regulated in T. brucei as in mammalian cells, to ensure sterol homeostasis.

Identification of a specific epitope on the extracellular domain of the LDL-receptor of Trypanosoma brucei brucei.

We have previously shown that an antiserum raised against the 86-kDa fragment of the low-density lipoprotein-receptor (LDL-receptor) of bloodstream Trypanosoma brucei brucei shows extensive cross-reactivity with the mammalian LDL-receptor. Here we report on the production and characterisation of 30 monoclonal antibodies (mAbs) raised against the same 86-kDa fragment of the T. b. brucei LDL-receptor. Of these, only 8 mAbs recognise in an ELISA test the purified (presumably intact) 145-kDa LDL-receptor. Seven of them also recognise the LDL-receptors isolated from rat and rabbit, whereas one mAb (1A9) is specific for the trypanosome LDL-receptor. A pool of several mAbs inhibits by 90% the specific binding of 125I-LDL to trypanosomes at 4 degrees C, but does not interfere with binding of 125I-LDL to rat fibroblasts. 125I-mAb 1A9 is efficiently taken up by T. b. brucei at 30 degrees C and its uptake is inhibited by an excess of unlabelled LDL particles, indicating that mAb 1A9 follows the LDL-receptor pathway. Uptake of 125I-mAb 1A9 by rat fibroblasts is less efficient and is not significantly reduced by an excess of unlabelled LDL. MAb 1A9 as well as other pooled mAbs activate rabbit complement, leading to lysis of trypanosomes in vitro. We conclude that the T. b. brucei LDL-receptor contains at least one specific epitope that is accessible on live cells to antibodies and which can activate the complement system.

Trypanosoma brucei brucei: antigenic stability of its LDL receptor and immunological cross-reactivity with the LDL receptor of the mammalian host.

The rapid growth of Trypanosoma brucei brucei in the blood and tissue fluids of vertebrates requires the receptor-mediated endocytosis of LDL from the host (Coppens et al. 1987; Gillett and Owen 1987) and is slowed by a monospecific rabbit antiserum against the purified LDL receptor of the parasite. We have used this antiserum in combination with several well-characterized antigenic variants (originating from stock 427: MITat 1.1a, 1.3a, 1.4a, 1.5a, 1.5d, 1.8b) to examine whether the LDL receptor of T. b. brucei is a stable surface antigen, common to all parasite variants despite antigenic variation of the major surface glycoprotein, and whether it is immunologically distinct from the LDL receptor of the host. At low concentrations, binding at 4 degrees C of rat LDL to several variants of T. b. brucei and to isolated rat hepatocytes was inhibited to a similar extent by the antiserum. In double immunodiffusion, a single precipitation line was observed, showing continuity between the extracts of all variants as well as between that of trypanosomes and of mammalian tissues. In Western blots of trypanosome extracts, the LDL receptor was strongly labeled as a single band of Mr 145,000, whereas with a rat liver extract, a single band of similar electrophoretic mobility was weakly labeled at a high concentration of the antiserum. In conclusion, the LDL receptor occurred in all variants of T. b. brucei, was a stable surface antigen despite variation of the major surface glycoprotein, and displayed biochemical and immunological similarities with the LDL receptor of the rat host.

A rapid method purifies a glycoprotein of Mr 145,000 as the LDL receptor of Trypanosoma brucei brucei.

The trypanosome LDL receptor has been isolated from bloodstream form and cultured insect-stage trypanosomes as a protein of Mr 145,000, using a rapid purification procedure in the presence of a cocktail of protease inhibitors, whereas previously a polypeptide of Mr 86,000 was purified as the LDL receptor. Both the 145,000 and the 86,000 polypeptides are glycosylated and recognized by a monospecific antibody raised against the 86,000 species. This antibody inhibits LDL binding to the intact trypanosomes, to the isolated 145,000 receptor and to the 86,000 species. Hence, the previously isolated 86,000 polypeptide is a degradation product probably representing the cleaved-off ectodomain of the trypanosome LDL receptor.