bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a dipeptidyl peptidase from Trypanosoma brucei
Authors: GIRARD D. , BASTOS I. M. D. , GRELLIER P.
Published: April 2005

Dipeptidyl peptidases (DPP) are ubiquitous enzymes. Several DPPs have been described and the most known among them is dipeptidyl peptidase IV, a type II multifunctional cell surface protein that belong to the prolyl oligopeptidase family of serine proteases. DPP IV specifically cleaves dipeptides from the N‐terminus of peptides with a Pro, Hyp or Ala in the penultimate position. DPP IV substrates include certain chemokines, growth factors (e.g. growth hormone, growth hormone‐releasing factor, glucagon‐like peptides 1 and 2) and neuro‐ and vasoactive peptides including neuropeptide Y, peptide YY and substance P. In this work, we present the identification of a dipeptidyl peptidase from Trypanosoma brucei (DPPTb). Its gene was isolated by PCR using primers designed from T. brucei ESTs sequences that matched with human DPP IV. However, the cloned and sequenced insert obtained displays similarity mainly with human DPP8 and DPP9. Active recombinant DPP Tb was expressed in insect cells using the baculovirus system. We observed by immunofluorescence, using anti‐DPPTb antibodies, that native DPPTb is localized in vesicles in the cytoplasm from procyclic forms of T. brucei.
Observation on a Enteromyxum leei (Myxozoa Myxosporea) Parasitosis on Farming Sea Bream Sparus aurata
Authors: JUBLANC E. , ELKIRIC N. , TOUBIANA M. , SRI WIDADA J. , LE BRETON A. , LEFEBVRE G. , SAUVEGRAIN C. , MARQUES A.
Published: April 2005

Enteromyxum leei parasitic infestation was first identified in 2001 on the site. Two years of investigations on this disease, when the infestation become detectable by light microscopic observation of the spores, it was observed that rearing parameters have very liitle influence on the evolution of the pathology, which then leads to heavy losses. This parasitic infestation is characterized by the location of the myxosporidia in the host (the digestive track), the absence of cyst formation and the occurrence of an amplification of the parasite development. PCR techniques allow to diagnose the first stages of the infestation on fish juvenile stages but have sofar not allowed the detection of a potential intermediate host. The epidemiological observations on site allowed recommending some actions to interfere with the life cycle of the parasite (cleaning of water channels should limit the prevalence of potential intermediate hosts) and adapting heath management to reduce the pressure of the pathogen (date of introduction of fingerlings on site, water temperature, oxygen level, flow rates). Two different hypotheses can explain the development of the disease in the present case on this site: (1) either a population of parasites in extension with a sexual reproduction and a complete life cycle on the site (2) or an asexual reproduction, such as a clone developing within the rearing structure and whose expansion may be related to transports of live fish and farming activities. The set up of health management procedures able to efficiently control the disease is strongly related to the understanding of this specific point.
Microsporidia of Fishes: Characterization of a New Species and Development of the Infectious Focus
Authors: MANSOUR L. , PRENSIER G. , BEN HASSINE O. K. , VIVARES C. P. , CORNILLOT E.
Published: April 2005

Fish parasites microsporidia are distributed among fifteen genera. In most cases, the infectious focus present a cyst‐like structure. According to the stage of evolution, the infection focus is either a xenoma or a granuloma. Specific host‐parasite interaction is at the origin of a large diversity of foci form and tissue specificity.

In this context, we describe Microgemma tincae n. sp. in the peacock wrasse, Symphodus tinca, fished near the Tunisian coasts. M. tincae is found in the liver of its host. Parasite characterization is based on the ultrastructural description of the life cycle (Transmission Electronic Microscopy) as well as on a part of the rDNA sequence (840 bp of the ssu gene). The inflammatory reaction responsible for the transition from xenoma to granuloma is carefully described. Prevalence is high and small fishes present high level of infection (several foci). One of the parameters responsible for the distribution of the number of foci per fish is the ratio between secondary and new infection.

We studied the Spraguea lophii infection to address this question at the molecular level in the angler fish, Lophius spp. We wanted to know if the reaction set up by the host was sufficient to stop the parasite propagation. The analysis was made in two‐dimensional (2D) PFGE at an infection focus scale (1‐mm3 samples). We compared 2D genome images from samples isolated from different parts of one or several infection foci of the same or different fish origin. We conclude that the fish immune system cannot avoid secondary infection.
Thymidine kinase and reverse genetics in Plasmodium falciparum
Authors: BONNEFOY S. , DIEZ M. , PENDYALA P. R. , BLANC C. , GUILLOTTE M. , MERCEREAU‐PUIJALON O.
Published: April 2005

In the past few years, genetic manipulation of Plasmodium falciparum has allowed functional studies of many parasite proteins. However, a careful interpretation of gene manipulation results is required, as chromosomal events may occur in vitro independently of the experimental modifications (gene invalidation, overexpression …), and because of the intrinsic mutagenic potential of genetic manipulation, or pleiotropic consequences related to the insertion of exogenous sequences. To ensure that mutant phenotypes arise from the planned mutations, it is necessary to obtain revertants re‐expressing the wild‐type gene. On this line, we have developed a positive/negative selection strategy allowing in a first step, gene targeting, then excision of the resistance marker. We converted the Toxoplasma gondii DHFR/TS into a tri‐functional enzyme following fusion with the Herpes virus type I thymidine kinase (HSV‐TK1). Positive selection corresponds to pyrimethamine resistant development, while negative selection acts in presence of the pro‐drogue ganciclovir. We used this tool to study RESA, a dense granule protein that associates with spectrin following invasion. Resa gene invalidation was obtained using a targeting plasmid allowing expression of the fusion DHFR/TS/HSV‐TK1, and molecular and phenotypic analyses of mutant parasites were performed. Next, wild‐type resa gene expression was restored after excision of all the exogenous sequences following phosphorylation of ganciclovir by the HSV thymidine kinase into a toxic metabolite. Phenotypic studies of revertants confirm some functions attributed to RESA protein, and validate the positive/negative strategy in this reverse genetic approach, a useful tool for development of “hit and run” mutagenesis in P. falciparum.
Intestinal microsporidiosis among HIV‐positive patients in Libreville (Gabon): Enterocytozoon bieneusi isolates show a very high diversity of genotypes
Authors: BRETON J. , KOMBILA M. , OKOME‐NKOUMOU M. , RINDER H. , THELLIER M.
Published: April 2005

Intestinal microsporidiosis caused by Enterocytozoon bieneusi and Encephalitozoon intestinalis are opportunistic infections occurring in severely immuno‐depressed patients, and may cause severe chronic diarrhea. Due to the extent of the HIV pandemic, microsporidia could pose a serious threat to public health in Africa. This study was conducted in Libreville, Gabon, central Africa, among HIV‐positive patients between September 2002 and December 2003. Criteria for inclusion were being HIV positive (HIV1 and/or HIV2), regardless of the CD4 cell count, stage of the disease or gastrointestinal symptoms. Nine hundred and ninety‐tree stool samples from 836 patients (mean age 38 years, range 18 months‐74 years) were screened for microsporidia by immunofluorescence using species‐specific monoclonal antibodies and confirmed by PCR. Twenty seven cases of E. bieneusi infection were found in the cohort, a prevalence of 2.7%. These patients were in the most severely immuno‐depressed group, 84% had a CD4 cell count below 200/mm3 compared to 57% of the cohort (mean=55 CD4+cells/mm3 compared to a mean of 88/mm3 for the microsporidia negative patients with less than 200 CD4/mm3).

The ribosomal DNA ITS sequence has been determined for several E. bieneusi isolates. A very high diversity of genotypes wasfound: eight different sequences were found from only 12 samples. This is surprisingly high since in previous studies conducted in Europe and America only 15 of the 49 E. bieneusi genotypes described have been found to infect humans. Only three genotypes from our samples have already been described: type K (two specimens). previously reported in human and cat, type D (one specimen) in human, pig and rhesus monkey, type E (1 specimen) in man, pig, racoon and muskrat. The five other genotypes are new and have been named Gabon‐1 to 5. The phylogenetic analysis of the sequences shows that genotypes Gabon‐1,2,3 and 4 (one specimen each) are close to types E, WL12, D and K, respectively, which belong to a group comprising most genotypes described so far. However, the Gabon‐5 genotype (four specimens) is only distantly related to this group and is most apparent to the group of genotypes (WL1‐3) recently described from racoons in North America. It was thus suggested that this group could represent another species or subspecies of E. bieneusi which are host‐adapted and do not infect humans. However, the finding of genotype Gabon‐5 shows that even distantly related genotypes infect severely immuno‐depressed humans.

This work was supported by the French National Agency for AIDS Research (ANRS‐1264).
Proteomics of the Microsporidia Encephalitozoon cuniculi: from Proteins of unknown Function to New Cell Wall Ones
Authors: BROSSON D. , TEXIER C. , PRENSIER G. , VIVARES C. P.
Published: April 2005

The microsporidia Encephalitozoon cuniculi, a human opportunist pathogen, is an obligate intracellular parasite, the nuclear genome of which has been fully sequenced. Our descriptive proteomic analysis of the late developmental stages showed that one‐third of the most abundant proteins are of unknown function. The spore, stage of resistance and dissemination, is protected by a wall, which plays the major role for survival in the environment. In order to find new spore wall candidates among the unknown proteins, a bioinformatic screening on the basis of the prediction of secretion, membrane anchoring and eventually of some domain homology was performed. In a first validation test, we used proteomics data to identify the target of a polyclonal antibody directed against the cell wall. The identified protein matched with criteria of selection and presented homologies with the polysaccharide deacetylase family, suggesting its implication in cell wall biogenesis. Its localization and the timing of its expression were studied by TEM immunocytochemistry. Screening was then applied to 44 expressed proteins of unknown function. Four of the nine potential cell wall components were demonstrated as being either endospore or exospore proteins, using polyclonal antibodies directed against GST recombinant proteins. The analysis of these new wall proteins should facilitate further bioinformatic screenings and should help in the better understanding of the structure and function of the microsporidian spore wall.
Plasmodium falciparum Genes Differentially Expressed During Merozoite Morphogenesis
Authors: CHARNEAU S. , FLORENT I. , GRELLIER P.
Published: April 2005

While the Plasmodium falciparum fully annotated genome has now been released, functional analysis of its expected about 5300 genes remains to be carried out to understand their roles during the parasite development, in particular for about 60% of genes devoid of significant similarities in other species. Here, a suppression subtractive hybridisation (SSH) screen was used to identify genes coding for proteins expressed during merozoite morphogenesis. The resulting library yielded clones with cDNA inserts ranging from a few hundreds to about a thousand base pairs. We report here the analysis of 50 independent cDNA clones and of the corresponding 40 P. falciparum genes. While 27.5% of them turned out to be previously described genes, all known to code for proteins expressed in schizont/merozoite stages (EBA‐175, MSP‐1, MSP‐3, MSP‐6, ABRA/MSP‐9, coronin, myosin A and the CLAG/RhopH1 protein), the remaining 72.5% were “putative” and “hypothetical” genes, coding for a variety of proteins among which we found several protease orthologs and orthologs of cytoskeleton‐associated and/or actin‐interacting proteins such as dynamin and formin‐2 as well as other rhoptry proteins. In addition to providing experimental evidence for their expression in schizont/merozoite stages, we discuss how these genes represent original candidates to investigate the molecular events required to elaborate infective merozoites.
Effect of the Disruption of RNA Pol II on the Transcription by RNA Pol l by Trypanosoma brucei
Authors: DEVAUX S. , LECORDIER L. , VANHAMME L. , PAYS E.
Published: April 2005

Trypanosomes exist in two major distinct forms: the procyclic form in insects in which the surface molecule is procyclin and the bloodstream form in mammals in which the surface molecule is VSG. The promoter of these locus recruits RNA Pol I. Despite the use of this polymerase, the transcripts are spliced and polyadenylated exactly as the mRNAs synthesised by RNA Pol II. The fact that mRNA production from these sites must involve a concerted action between RNA Pol I and a “RNA factory” normally linked to RNA Pol II, led to the proposal that the control of these Pol I transcription units would involve a limiting component bridging RNA Pol I and an elongation/processing complex normally associated with RNA Pol II. To study the effects of the disruption of RNA Pol II on VSG expression level, we cloned the T. brucei homologue of the rpb9 subunit of RNA Pol II, which plays a role in RNA elongation in other eukaryotic cells. In PF, disruption of Tbrpb9 by conditional RNAi led to a strong transcriptional stimulation in the beginning of the ESs. This effect was linked to the inhibition of procyclin transcription. Thus, the disruption of RNA Pol II abolished the stage‐specific regulation of the transcription units for the two major surface antigens, both of which recruit RNA Pol I.
Revisiting Putative Functional Properties of the Plasmodium falciparum Pf155/RESA Protein Using Genetically Engineered Parasites
Authors: DIEZ M. , GUILLOTTE M. , LE SCANF C. , CONTAMIN H. , DAVID P. , COOKE B. , MERCERAU‐PUIJALON O. , BONNEFOY S.
Published: April 2005

Pf155/RESA (Ring Infected Erythrocyte Surface Antigen), is a Plasmodium falciparum protein discharged by the dense granules of merozoites during invasion and exported to the inner face of the erythrocyte membrane, where it interacts with spectrin in the young stages. Chromosome 1 subtelomeric deletion, eliminating amongst others the resa gene, may occur during adaptation of parasite isolates to in vitro culture. This is accompanied by erythrocyte membrane modifications of the red blood cell, such as increased adhesion and effect on membrane rigidity. It has been proposed that RESA was likely to contribute to these functional and rheological modifications. To assess this, we have constructed resa knock‐out parasites in the FUP/CB strain. This results in negativating the erythrocyte membrane immunofluorescence of glutaraldehyde‐fixed red blood cells (EMIF), indicating that RESA is critical for EMIF reactivity of hyperimmune human sera. Phenotypic and functional analysis of resa k.o. parasites indicated that loss of RESA expression neither affects membrane rigidity nor CD36 binding under flow conditions. Furthermore, infection of Saimiri sciureus monkeys showed that resa gene deletion does not account on its own for the greater adaptation and parasite virulence in this model.
Paramyxea in Scandinavia
Authors: LARSSON J. I. R. , KØIE M.
Published: April 2005

Paramyxea is a small phylum of marine parasites, using molluscs, crustaceans or polychaetes as hosts. Probably the group is cosmopolitan, but the distribution known so fais restricted to southern Europe, Australia and East Asia. Polychaetes of the genus Nephtys, collected from Danish waters, are infested with a paramyxean parasite in the gut epithelium. The parasite develops intracellularly and is in close contact with the cytoplasm of the host cell. When harbouring mature spores gut cells are lysed and spores are released into the digestive tract.

Reproduction is by endogeneous budding, typical for Paramyxea, and the only reproduction observed is sporogony. A stem cell (cell 1) yields two or four sporonts (cell 2), each of which produces four tetracellular spores (cells 3–6). The mature spore is fusiform, about 10 microns long, and surrounded by an individual sac filled with fibrous material. The sacs are united to a spherial body with prominent striated terminal projections. Haplosporosomes, of three different types, are present in cells 1, 4 and 5. The species, which is new to science, is the first find of Paramyxea from Scandinavia.
Protozoan population and fermentation parameters of rumen contents of sheep from heavy metal contaminated area
Authors: VÁRADYOVÁ Z. , MIHALIKOVÁ K. , JAVORSKY P. , KISIDAYOVÁ S.
Published: April 2005

The rumen contents from Slovak Merino sheep (12 months of age) were used for in vitro study on the influence of area contaminated by heavy metals on rumen fermentation and protozoan activity. Sheep were browsing in the contaminated area of Kal'ava village (Slovakia) and were exposed to 1‐year intake of heavy metals. The area of Kal'ava is contaminated by atmospheric pollution from the non‐ferrous metal works at Krompachy (Slovakia). Based on the levels of mercury (4.8 mg'kg), copper (232.9 mg/kg), cadmium (1.2 mg/kg), lead (92.5 mg/kg) and arsenic (74.6 mg/1) the soil was categorized as profusely contaminated. Grass contamination was below the toxic limits. In the tested materials, copper was present at the highest levels followed by lead and arsenic. Meadow hay was used as a tested substrate of fermentation activity; it was incubated with buffered rumen fluid for 24 h. The significantly decreased values of fermentation parameters (total gas, methane, total VFA, acetate) were associated with a reduced total concentration of protozoans. Significant decrease was detected in the total rumen ciliate population and population of c. No significant differences were observed in the concentration of Dasytricha ruminantium and Ophryoscolex c. tricoronatus.

The study was supported by funds from Grant Agency for Science of the Slovak Academy of Sciences (2/3058/23, 2/3064/23) and funds from APVT grant (51012602).
Role of IscS in Fe‐S cluster assembly in Trypanosoma brucei
Authors: SMÍD O. , VONDRUSKOVÁ E. , VILÍMOVÁ V. , SUT′ÁK R. , LUKE J. , TACHEZY J.
Published: April 2005

Despite the significance of proteins containing iron‐sulfur cluster (Fe–S proteins), the processes of Fe–S cluster assembly and maturation of Fe–S proteins are poorly understood. However, several key proteins involved in the assembly have been identified, notably IscS, a cystein desulfurase, which provides sulfur for Fe–S cluster and IscU, a metallochaperone acting as a scaffold for cluster assembly. In this work, we studied the process of Fe–S cluster biosynthesis in Trypanosoma brucei by identifying the homologue of IscS in the T. brucei (TbIscS). To address the function of TbIscS, we inhibited its expression by means of RNA interference (RNAi). After RNAi induction, generation time of the TbIscS knock‐down cell line was significantly prolonged. All types of mitochondrial ATP production in the cells were severely affected. Analysis of glucose metabolism end products determined pyruvate as major excreted metabolite of the induced cells, while the uninduced cells produced only small amount of this glycolytic end product. These data demonstrate that mitochondrial metabolism is impaired in cells with TbIscS knocked down. To test whether the observed phenomena were results of Fe–S cluster assembly disruption, we examined the Fe–S cluster‐dependent activity of aconitase. This enzyme is localized in its active form in mitochondrion as well as in cytosol of T. brucei. After RNAi induction we observed the reduction of aconitase activity in both compartments (approx. 70% reduction in cytosol, approx. 30% in mitochondria). Western blots together with the EPR analysis showed that the reduction in cytosolic activity was due to impaired Fe–S cluster formation, while decrease in aconitase activity in mitochondria corresponded to the reduced level of the protein.
Parasitic Stramenopila
Authors: KOSTKA M. , HAMPL V. , CEPICKA I. , FLEGR J.
Published: April 2005

In the vast and diversified eukaryotic group Stramenopila we can find many different life strategies, one of them being parasitism. There are two groups of stramenopiles containing important parasites of vertebrates: the oomycetes (e.g. Saprolegnia) and the wholly parasitic group Slopalinida+its sister taxon—Blastocystis. The order Slopalinida comprises families Opalinidae and Proteromonadidae. The two families are considered related based on the structure of basal bodies and their appendages and the presence of subpelicular microtubules. However, a robust recognition of phylogenetic affinities of Opalinidae—the peculiar multinucleated intestine commensals of frogs—has been hindered by the absence of reliable molecular data. Up to now, all attempts to sequence opalinid genes failed, as the obtained sequences labeled as Protoopalina intestinalis, Cepedea virguloidea and Opalina ranarum in GenBank apparently originate from a zygomycete contamination. We present the first molecular data for the family Opalinidae—SSU rRNA gene of Protoopalina intestinalis. Our phylogenetic analyses undoubtedly show opalinids as a sister group to Proteromonas within the Stramenopila clade, confirming the monophyly of Patterson's order Slopalinida. The enigmatic genus Blastocystis is resolved with high statistical support as a sister group to Slopalinida. Our analyses clearly demonstrate that Cavalier–Smith's phylum Bigyra, which comprises Oomycetes and their relatives together with Slopalinida and Blastocystis, is not monophyletic. We also show, that Blastocystis sp. isolate obtained from the red‐footed tortoise (Geochelone carbonaria), forms a sister group to all Blastocystis isolates from birds and mammals sequenced so far.
A multidisciplinary approach to describe protists: redescription of Chattonidium setense Villenueve, 1937 (Ciliophora, Heterotrichea)
Authors: MODEO LETIZIA , PETRONI GIULIO , VERNI FRANCO , ROSATI GIOVANNA
Published: April 2005

This study represents the first extended report on the heterotrichid ciliate Chattonidium setense since the original description by Villeneuve in 1937. This rare marine ciliate is in fact the only representative of the family Chattonidiidae, which is the eighth, often neglected, family of order Heterotrichida. As in our previous works, here we redescribe the species combining morphological observations (live, stained, scanning, and transmission electron microscope) with behavioral and molecular data as we are convinced that both taxonomic determination and molecular characterization are necessary for the clarification of phylogenetic relationships between a species and related genera. Live observation, Feulgen procedure and SEM analysis showed, besides a number of other interesting features, the presence of a characteristic aboral ciliated cavity of undetermined function, whose presence makes ciliate's identification unambiguous. TEM observation evidenced the following main features: well‐divided ecto‐and endoplasm; deep insertions of the rows of somatic dikinetids in cortex invaginations; paraglycogen granules as polysaccharide reserve; large bundles of microtubules and fibers delimiting the body region surrounding the aboral cavity; two kinds of subcortical extrusomes. Since the revision of heterotrichs ciliates by Villeneuve‐Brachon (1940) many genera and families have been transferred to other classes on the basis of both new molecular data and reconsidered morphological characters. Our results support previous statements that Chattonidium has the general morphology typical of Heterotrichida and that the main similarities are with genus Condylostoma. 18S rRNA molecular data firmly confirm that Chattonidium belongs to class Hetrotrichea, order Hetrotrichida (Lynn, D. H. 2003. The Ciliate Resource Archive. http://www.uoguelph.ca/~ciliates2004.07.19) as an independent lineage. Preliminary molecular data also suggest an association between genera Chattonidium and Condylostoma. Finally, some behavioral and ecological notes on the ciliate are added.
Effect of euplotin C on swimming behavior in Paramecium
Authors: RAMOINO PAOLA , USAI CESARE , GUELLA GRAZIANO , DINI FERNANDO
Published: April 2005

The effect of euplotin C—a lipophilic toxic metabolite produced by the protist ciliate Euplotes crassus—on the swimming behavior was studied in a single‐celled system represented by the ciliate Paramecium primaurelia. When in P. primaurelia internal Ca2+ concentration coupled to membrane depolarization increases, a reversal in the direction of ciliary beating and consequently in swimming direction occurs. The ciliary reversal is correlated to Ca2+ influx amount. In this study, evidence was given that continuous ciliary reversal (CCR) duration, induced by high external KCl concentrations, is longer in euplotin C‐treated cells than in control cells. To test the hypothesis that euplotin C increases CCR duration by modulating a specific subtype of Ca2+ channel, selective Ca2+ channel blockers were used. Blocking L‐type channels by nimodipine and verapamil, N‐ and Q‐type channels by Ω‐conotoxins, fractions GVIA and MVIIC, significantly reduced the CCR duration evoked by membrane depolarization, suggesting an involvement of these channels in ciliary reversal in Paramecium. The effect of euplotin C on CCR duration persisted when Ω‐conotoxin GVIA or Ω‐conotoxin MVIIC were applied, conversely, it disappeared when L‐type channel blockers were used. The magnitude of the block by nimodipine and verapamil in the presence of euplotin C was comparable with that observed in the absence of euplotin C, suggesting that the Ca2+ channels modulated by euplotin C were dihydropyridine‐sensible calcium channels similar to L‐type channels found in mammalian cells.
Activity of a killer peptide on the growth and ultrastructure of leishmaniae
Authors: SCUTERA SARA , RAIMONDO STEFANIA , CONTI STEFANIA , MAGLIANI WALTER , POLONELLI LUCIANO , SAVOIA DIANELLA
Published: April 2005

An engineered killer decapeptide (KP) has been recently synthesized based on the sequence of a recombinant, single‐chain anti‐idiotypic antibody acting as a functional internal image of a yeast killer toxin. This KP exerts a microbicidal activity by an interaction with β‐glucan present in the surface of many microorganisms, in particular fungi. The activity of KP against the “in vitro” growth and vitality of promastigotes of two Leishmania species has been evaluated and compared with the activity due to a “scramble” irrelevant decapeptide (SP). The IC50 of KP corresponds to 7.2 × 10−5mol/litre for Leishmania infantum, whereas both the inhibitory and leishmanicidal activity is superior on Leishmania major. Moreover, an increase of activity was observed by treating protozoa again with KP; SP is inactive. Structural damages of promastigotes were revealed by transmission electron microscopic studies; the treatment with KP induces a cell death via a non‐apoptotic process. In particular, intracellular damage including cytoplasmic degeneration without disruption of the plasma membrane was noted. The activity of KP on leishmania promastigotes seems governed by ionic and/or electrostatic interactions with the glycoconjugates present on the surface of the parasite inducing an authophagic cell degeneration.
Characterization of R‐body genetic determinants in Caedibacter caryophilus a symbiont of Paramecium caudatum: preliminary results
Authors: SCHRALLHAMMER MARTINA , GÖRTZ HANS‐DIETER , LUDWIG WOLFGANG , SCHLEIFER KARL‐HEINZ , PETRONI GIULIO
Published: April 2005

Refractile inclusion bodies, called R‐bodies were observed within the cells of some bacterial strains. They are protein ribbons, which are typically coiled into cylindrical structures. They are produced by members of the genus Caedibacter, gram‐negative rod‐shaped endosymbionts of paramecia and e.g. the free‐living bacteria Hydrogenophaga taeniospiralis and Acidovorax avenae. The phylogenetic relationship even between the members of the genus Caedibacter is quite low: C. taeniospiralis belongs to the Gammaproteobacteria and is related to Francisella tularensis, C. caryophilus is affiliated with the Alphaproteobacteria and clusters with the obligate endosymbiont Holospora. In the case of C. taeniospiralis 51, the genetic determinants of R‐body synthesis are located on a plasmid, whereas in other strains like 7 and 562 it looks like phage particles are involved in their production. In the present study, we investigated C. caryophilus, endosymbiont of Paramecium caudatum. Separation of C. caryophilus cells was performed by Percoll™ density gradient centrifugation. The isolated DNA was separated by pulsed‐field gel electrophoresis and it was possible to visualize several bands referring to one or more extrachromosomal elements. A small gene library of these extrachromosomal elements was constructed and we already identified transposition‐related genes; interestingly, similar genes were reported also on the plasmid of C. taeniospiralis 51.
An alternative approach for the molecular characterization of uncultivable protists retaining morphological information
Authors: ANDREOLI ILARIA , FERRANTINI FILIPPO , MANGINI LARA , SANTANGELO GIOVANNI , PETRONI GIULIO , VERNI FRANCO
Published: April 2005

Recent culture‐independent studies based on small‐subunit ribosomal RNA (SSU rRNA) gene analysis revealed the existence of completely new clades of protists. The main problems with this approach are to correlate sequences from environmental rRNA genes with the organisms they belong to and then to detect the ecological role of these organisms in the environment. In order to overcome such problems we chose an alternative approach allowing us both a molecular characterization of uncultivable organisms with a low relative abundance in environmental samples, and a morphological analysis, even if restricted. The experimental protocol consists of two steps: an initial observation and photo‐taking of the single cell under the DIC (Differential Interferential Contrast) microscope and then PCR amplification and direct sequencing of the 18S rRNA gene of the same cell. The advantages of this method are the possibility to: (1) establish a precise link between morphology and gene sequence; (2) detect the possible occurrence of highly similar species within the studied population; (3) avoid the insertion of Taq‐polymerase errors in the gene sequence; and (4) detect possible polymorphisms in the gene under examination. Such an approach was used to sequence the 18S rRNA gene of organisms belonging to the class Karyorelictea that comprises several uncultivable ciliates with limited distinctive features. Gene sequences analysis revealed an unexpected genetic variability in trachelocercids and, in particular, the existence of polymorphisms in the SSU rRNA gene of a group of them. Such specimens show a similar morphology and, as a result of phylogenetic analysis, they form a constant clade.
A non‐staining chlorhexidine mouthwash? Probably not: a study in vitro
Authors: Addy M , Sharif N , Moran J
Published: April 2005

Background and aim:  Tooth staining is a common side effect of chlorhexidine mouth rinses and caused by the interaction of the di‐cationic antiseptic with dietary chromogens. A product is now available, which claims an anti‐discolouration system (ADS) with one clinical study in support. This study in vitro aims to determine whether two ADS rinses do or do not bind dietary chromogens.

Method and materials:  Optically clear acrylic specimens were cycled through human saliva (2 min), one of the three chlorhexidine rinses (two ADS and a positive control) (2 min) or water and then soaked in tea (60 min). After each cycle the optical density (OD) of specimens were read on a UV/visible spectrophotometer. The exit point was the cycle at which OD was >2.0.

Results:  All three rinses exceeded OD 2 at 11 cycles and there was no significant difference in staining for the ADS rinses compared with the positive control rinse.

Conclusion:  Based on extensive literature for the correlation of this test in vitro with chlorhexidine anti‐plaque activity and propensity to stain in vivo these ADS rinses will have the same anti‐plaque efficacy and potential to cause stain as established chlorhexidine rinse products.
Dental anxiety in children and its relationship to dental caries and gingival condition
Authors: Taani DQ , El‐Qaderi SS , Abu Alhaija ESJ
Published: April 2005

The aim of this study was to determine the levels of dental anxiety, dental caries and gingivitis among 12–15‐year‐old schoolchildren, in Irbid Governorate/northern Jordan, and to evaluate the correlation between these variables. Two schools were selected by a simple random method from each of the five geographic areas in Irbid Governorate. All children (1021), from the 10 selected schools, who participated in this study completed a questionnaire modified from Kleinknecht's Dental Fear Survey (DFS questionnaire). Children underwent oral examination for dental caries and gingival condition, using Decayed, Missing and Filled Teeth (DMFT) Index and Löe and Silness Gingival Index (GI), respectively. Results of this survey showed that the prevalence of low to moderate ‘general dental fear’ among the study population was 43% while that of ‘high dental fear’ was 10%. The self‐reported ‘general fear of dental treatment’ was higher among girls than boys. Fear of specific stimuli (pain) was the most common source of dental fear. The sight and sensation of the anaesthetic needle and the sight, sound and sensation of the drill were rated the most fear‐eliciting stimuli. The mean DMFT (2.89) and GI (1.80) of boys was not significantly different from the DMFT (3.37) and GI (1.53) of girls (P > 0.05). Spearman's correlation test demonstrated no association between ‘general dental fear’ and dental caries (r = 0.06) or gingivitis (r = 0.007).
Work stress and burnout among dental hygienists
Authors: Gorter RC
Published: April 2005

Objectives:  The aim of this study was to describe the factors associated with work stress and burnout among dental hygienists. Furthermore, how to deal with work stressors in order to prevent burnout in the dental hygienist work environment will be discussed.

Methods:  From a small literature search, only a few empirical studies could be traced that describe work stress or burnout among dental hygienists.

Results:  Burnout incidence among dental hygienists appears to be relatively favourable, when compared with other professions, according to a 20‐year‐old study. On the contrary, in a recent study, it was described that one out of eight dental hygienists felt emotionally exhausted from work. Dental hygienists, when compared with other professionals, are relatively negative about the variety of tasks they find in their work. Factors associated with experienced work stress are, according to another study, musculoskeletal pain, combining work and private life, highly efficient organization of work, long working hours, working without assistant, difficult or demanding patients, lack of leisure time, lack of support by practice management and doubts about one's own capabilities. Some factors that may prevent burnout are: recognition of one's own work pressure, learning to unwind, time management and organization of work, realistic career expectancies, social skills, healthy life‐style, peer‐group contacts and pre‐graduate reflection.

Conclusions:  There appears to be a lack of recent data on burnout among dental hygienists. Although some knowledge exists on work stressors, a thorough investigation on burnout incidence, risk factors, as well as job resources is needed.
Tooth loss among habitual chewing‐stick and plastic toothbrush users in the adult population of Mtwara, rural Tanzania1
Authors: Mumghamba EGS , Fabian FM
Published: April 2005

Objectives:  The aim of this study was to determine the extent and type of tooth loss in relation to habitual chewing‐stick (CS) and plastic toothbrush (PT) users in adults.

Methods:  A cross‐sectional descriptive study was conducted in Mtwara's rural population aged ≥40 years. A total of 206 randomly selected study participants – males (55.8%) and females (44.2%) – were interviewed on oral hygiene practices using a structured questionnaire, and a clinical examination performed for assessment of tooth loss.

Results:  Of the total, 93.2% were partially edentulous, 2.4% completely edentulous and those with no tooth loss 4.4%. The total number of missing teeth were 1728 (mean, 8.38 ± 7.9), and the causes were caries (74.9%), mutilation (4.8%) and others including periodontal and trauma (20.3%). There was no significant difference in the mean number of tooth loss between the upper and lower jaw, and between males and females. However, the mean number of missing teeth in the lower jaw was higher in females than in males (P = 0.04), and on lower left than lower right jaw (P = 0.008). Lower molars, especially first molars, were the teeth lost most often. Tooth‐cleaning devices included PT (51.5%), CS (25.7%), both type (BT; 17.0%) and unspecified (5.8%). The differences in the mean number of missing teeth were higher in CS than habitual PT users (P = 0.024) and in BT (P = 0.029). Tooth loss was also higher among those who brushed once when compared with those who brushed two times or more per day (P = 0.046).

Conclusion:  Tooth loss was significantly higher in CS than PT habitual users and affected mostly the lower molars and especially the first molar. However, for economical reasons, effective use of CS should be promoted for enhanced tooth retention.
GOLBALIZATION, CORPORATE FINANCE, AND THE COST OF CAPITAL
Authors: Stulz René M.
Published: April 2005

International financial markets are progressively becoming one huge, integrated, global capital market—a development that is contributing to higher stock prices in developed as well as developing economies. For companies that are large and visible enough to attract global investors, having a global shareholder base means having a lower cost of capital and hence a greater equity value for two main reasons:

First, because the risks of equity are shared among more investors with different portfolio exposures and hence a different “appetite” for bearing certain risks, equity market risk premiums should fall for all companies in countries with access to global markets. Although the largest reductions in cost of capital resulting from globalization will be experienced by companies in liberalizing economies that are gaining access to the global markets for the first time, risk premiums can also be expected to fall for firms in long‐integrated markets as well.

Second, when firms in countries with less‐developed capital markets raise capital in the public markets of countries (like the U.S.) with highly developed markets, they get more than lower‐cost capital; they also import at least aspects of the corporate governance systems that prevail in those markets. For companies accustomed to less‐developed markets, raising capital overseas is likely to mean that more sophisticated investors, armed with more advanced technologies, will participate in monitoring their performance and management. And, in a virtuous cycle, more effective monitoring increases investor confidence in the future performance of those companies and so improves the terms on which they raise capital.

Besides reducing market risk premiums and improving corporate governance, globalization also affects the systematic risk, or “beta,” of individual companies. In global markets, the beta of a firm's equity depends on how the stock contributes to the volatility not of the home market portfolio, but of the world market portfolio. For companies with access to global capital markets whose profitability is tied more closely to the local than to the global economy, use of the traditional Capital Asset Pricing Model (CAPM) will overstate the cost of capital because risks that are not diversifiable within a national economy can be diversified by holding a global portfolio. Thus, to reflect the new reality of a globally determined cost of capital, all companies with access to global markets should consider using a global CAPM that views a company as part of the global portfolio of stocks. In making this argument, the article reviews the growing body of academic studies that provide evidence of the predictive power of the global CAPM as well as the reduction in world risk premiums.
PETROZUATA: A CASE STUDY OF THE EFFECTIVE USE OF PROJECT FINANCE
Authors: Estry Benjamin C.
Published: April 2005

Companies are increasingly using project finance to fund large‐scale capital expenditures. In fact, private companies invested $96 billion in project finance deals in 1998, down from $119 billion in 1997 largely due to the Asian crisis, but up more than threefold since 1994.

The decision to use project finance involves an explicit choice of organizational form as well as financial structure. With project finance, sponsoring firms create legally distinct entities to develop, manage, and finance the project. These entities borrow on a limited or non‐recourse basis, which means that loan repayment depends on the project's cash flows rather than on the assets or general credit of the sponsoring organizations. Despite the non‐recourse nature of project borrowing, projects are highly leveraged entities, with debt to total capitalization ratios averaging 60–70%.

Petrozuata, a $2.4 billion oil field development project in Venezuela, is a recent example of the effective use of project finance for several reasons. First, the analysis shows a typical setting where project finance is likely to create value, that of a large‐scale investment in Greenfield assets (in this case, wells, pipelines, and upgrader) that can function as a stand‐alone economic entity and support a high leverage ratio. Given the nature of this investment, one can think of project finance as venture capital for fixed assets, except that the investments are 100 to 1000 times larger and financed primarily with debt rather than equity.

Besides highlighting the types of assets appropriate for project finance, this article illustrates the sizeable transactions costs associated with structuring a deal as well as the full range of benefits accruing to project sponsors. The structure allows sponsors to capture tax benefits not otherwise available, reduces information costs for creditors and other investors, and lowers the overall cost of financial distress. The combination of high leverage, concentrated equity ownership, and direct control in project finance also addresses a wide range of incentive problems that destroy value in diversified companies.

Analysis of the explicit contractual terms of the deal reveals a careful allocation of project risks in an attempt to elicit optimal behavior by each of the participants. As illustrated in the Petrozuata case, limiting completion and operating risks are important undertakings. But project finance is most valuable as an instrument for managing sovereign risks. Indeed, the ability of project finance to limit sovereign risk is the one feature that cannot be replicated under conventional corporate financing schemes.
STRATEGIC HEDGING
Authors: Mello Antonio S. , Parsons John E.
Published: April 2005

This article presents a new approach to financial risk management whose primary objective is to ensure that companies have sufficient internal funds and access to outside capital to carry out their strategic investments. The foundation of this approach is a comprehensive measure of corporate exposure that views the firm as a collection of current cashgenerating assets and future investment opportunities and that attempts to show how changes in fundamental economic variables can threaten the firm's ability to realize its strategic objectives. As such, the measure of exposure reflects the effect of expected changes in economic variables not only on the firm's operating cash flows but also on its future investment requirements.

Because its focuses only on the exposures that need protection when regular sources of funds are exhausted, this strategic hedging approach will generally lead to a more conservative hedging policy. In so doing, it should enable companies to avoid the excessive and costly “micro” hedging of individual transactions—an approach that can easily degenerate into speculation.
MEASURING THE COST OF CAPITAL IN AN INTERNATIONAL CAPM FRAMEWORK
Authors: Schramn Ronald M. , Wang Henry N.
Published: April 2005

This paper can be viewed as extending the traditional CAPM framework in two important ways. The first expands the concept of the market portfolio to include international securities. The second extends the definition of systematic risk to include currency risk.

What becomes clear in estimating the cost of capital for an international asset is that both extensions have become necessary if the traditional CAPM is to remain relevant. International markets have become increasingly integrated over the past two decades and so all assets might now be considered “international” and priced accordingly. The inclusion of the currency risk factor is not an ad hoc addition to the CAPM but rather results quite naturally from the fact that foreign returns need to be converted into a domestic currency.

Based on an examination of 18 companies, the article shows that the use of a broader market portfolio will tend to lower the estimated cost of capital for most firms, but in some cases could actually raise it. (In the case of Singapore Airlines, for example, the currency risk factor adds substantially to the cost of capital, while materially reducing it in the case of Nestlé.) Using a simple regression, the authors also attempt to show how the specifics of a particular company—for example, the currencies that are part of their cost/ revenue structure—determine the impact of currency risk on the cost of capital.
Are Banks Still Special? New Evidence on Their Role in the Corporate Capital‐Raising Process
Authors: James Christopher , Smith David C.
Published: April 2005

Bankers appear to play a special role in providing commitment‐based financing to corporations. This type of lending is important not only for small firms that lack access to public debt markets but for large and medium‐size companies as well. For such companies, commitment‐based financing provides access to debt capital that becomes valuable when the firm has an immediate need for funding but interest rates in public debt markets are prohibitively high, or the firm is undervalued by the market. A good example of this was provided by the Asian crisis in the last quarter of 1998, when $10 billion of commercial paper was retired and $20 billion of net new commercial loans were booked. The authors also suggest that the fact that commitment‐based financing is used by larger companies when they believe themselves to be undervalued in the market is probably the best explanation of why announcements of these types of loans elicit a positive stock price reaction.