bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth of the vacuoleless mutant of Tetrahymena thermophila NP1 in phytate
Authors: WEBB SAMANTHA , SMITH‐SOMERVILLE HARRIETT E.
Published: April 2005

Phytate, the salt form of phytic acid, is the major store of phosphate in seeds and grain. Since non‐ruminant farm animals poorly digest phytate, it is also a source of environmental phosphate contamination in agricultural areas. We are using Tetrahymena, a ciliated protist with multiple routes for nutrient assimilation, as a model to investigate the contribution of heterotrophic protists to the environmental cycling of phosphate from phytate. This ciliate has the ability to grow on phytate as the sole phosphate source (Ziemkiewicz, H. T., Johnson, M. D. & Smith‐Somerville, H. E. 2002. J. Eukaryot. Microbiol., 49:428). Tetrahymena thermophila NP1, a temperature‐sensitive vacuoleless mutant (ATCC #50202), provides a way to separate membrane transport from uptake through phagosomes, and to assess the importance of each mechanism. This cell grows equally well at the permissive and non‐permissive temperatures with either phytate or inorganic phosphate as the phosphate source. Our results demonstrate that phagosomes are not required to use the phosphate from phytate.
A Sec7‐related Protein in Tetrahymena
Authors: BELL AARON J. , GUERRA CHARLES F. , SATIR PETER
Published: April 2005

Sec7 was first identified in mutants of Saccharomyces cerevisiae that accumulated Golgi stacks and cisternae when incubated at 37°C in low glucose medium. Since then, Sec7‐related proteins have been found in a variety of organisms including Homo sapiens, Arabidopsis and Paramecium. In their role in vesicular transport, many of the Sec7‐related proteins have been shown to function as guanine‐nucleotide exchange factors (GEFs) for small G‐proteins such as ARF. Paramecium sec7 (psec7) has been cloned and shown to be up‐regulated upon deciliation, implicating a role of pSec7 in ciliogenesis and/or ciliary function. The presence of a Sec7‐related protein in Tetrahymena thermophila has been shown by immunoblotting and immunofluorescence using pSec7 antibodies against T. thermophila proteins. These experiments confirm the localization of tSec7 to the cilia and ciliary membranes of Tetrahymena. These results suggest that tSec7, like pSec7, is a ciliary‐membrane‐associated protein that could participate in signal transduction processes. The genomic sequence of tsec7 is now known. RT‐PCR has revealed the presence of 9 introns within tsec7, the largest of which is 327 bps in length. Predicted start and stop codons within the genomic sequence will be verified with mass spectroscopic analysis of the protein. Alignment of pSec7 and tSec7 amino acid sequences indicates a 32% identity and a 52% homology between the two proteins. In addition, motifs found in pSec7 (i.e. the Sec7 domain, truncated IQ motifs and PH domains) are also found in tSec7.
Trichomonas and GFP, the absolute misfit …?
Authors: BRICHEUX G. , COFFE G.
Published: April 2005

Trichomonas vaginalis belongs to a group of parasitic protists with a significant impact on human health, such as Giardia duodenalis and Entamoeba histolytica. This microorganism is able to assume a dynamical behavior towards its host; in contact with the vaginal epithelium cells, the swimming form of Trichomonas modifies its sub‐membraneous cytoskeleton, and changes to an amoeboid and adherent form to the host cells. Using this adherence mechanism, Trichomonas is able to invade the vaginal mucous membrane while remaining an extracellular parasite. Thus, there is a direct link between cytoskeletal dynamics and pathogenicity. We have characterized some of the cytoskeleal proteins from Trichomonas: actin, α‐actinine, coronin, and more recently gelsolin. Also, the genome sequencing of this parasite is under way, and will bring numerous data, particularly putative proteins that will need further functional identification. We are presently working to tune up some in vivo analysis methods in order to study these new proteins. The use of the GFP (green fluorescent protein) as a tag to study gene expression and protein localization becomes more and more widely reported. However, this method requires precise minimal conditions in order to satisfy the physico‐chemical constraints of the chromophore and in the same time to respect the physiology of the eukaryotic cell.
A new real‐time RT‐PCR method allowing reliable absolute quantification of human cytokine mRNA in paediatric malaria clinical samples
Authors: BOEUF P. , VIGAN I. , JUBLOT D. , BARALE J.‐C. , GOKA B. , KURTZHALS J. , AKANMORI B. D. , HVIID L. , PUIJALON O. , BEHR C.
Published: April 2005

In endemic areas, clinical manifestations of Plasmodium falciparum infection range from asymptomatic parasitaemia to life‐threatening severe syndromes. Immune differences that could account for this disparity are poorly understood. Using tight criteria to classify patients into non‐overlapping clinical categories, we showed that cerebral malaria and severe anaemia were distinct immunological syndromes and that a proper quantitative description of cytokine profiles in the various clinical groups is essential to the understanding of the activation of immunocompetent cells.

Due to the limited size of paediatric blood samples, we chose to measure cytokine mRNA using real‐time RT‐PCR. We showed that RT efficiency displayed intra‐and intergenic variations that have to be taken into consideration for reliable absolute RNA quantification when comparing clinical cases. We thus developed a SYBR Green I‐based real‐time RT‐PCR method using synthetic external RNA standards specific for each gene. Absolute RNA quantification is achieved by reverse transcribing known copy numbers of this RNA standard in parallel with cellular RNA. Strictly specific primers were designed to allow the quantification of any RNA in the same thermocycling parameters for future automation. Our method gave similar results for a lower cost when compared with TaqMan, and led to reproducible and reliable absolute RNA quantification. We validated it in vitro on naïve PBMC stimulated by LPS and ex vivo on PBMC from malaria patients. This new method raises the unprecedented possibility to compare cytokine mRNA levels between different clinical groups and is a powerful tool to further study the inflammation processes associated to malaria.
Preservation of ptp1 and ptp2 Gene Synteny in Two Microsporidia Parasite of Insects: Nosema locustae and Nosema grylli
Authors: POLONAIS V. , DELBAC F. , VIVARES C. P.
Published: April 2005

Microsporidia are obligate intracellular parasites infecting a wide range of invertebrate and vertebrate hosts including human. The spore contains a specialized organelle called the polar tube involved in host cell invasion. Three polar tube proteins (PTP1‐3) have been identified in Encephalitozoon cuniculi. The genes coding for ptp1 and ptp2 are localized on the same chromosome and are at a distance of 860 bp. A similar synteny organization is found in two other Encephalitozoon species. Comparison of the E. cuniculi genome with that of Nosema locustae, an microsporidia parasite of grasshoppers, indicates that several genes are positioned in the same order and orientation. This allowed the identification of two ORFs distant of 1117 bp, that despite divergent sequence features, encode proteins having common characteristics with Encephalitozoon PTP1 and PTP2 (signal peptide, proline‐rich internal repeats for PTP1, lysine‐rich protein for PTP2 …). To test whether they correspond to PTP, polyclonal sera were raised against two recombinant proteins expressed in E. coli. In indirect immunofluorescence, the extruded polar tubes of N. locustae spores are specifically labelled. NlPTP1 and NlPTP2 have 355 and 287 amino acids, respectively, and are only soluble in the presence of high concentrations of reducing agent.

Aptp1 and ptp2 containing cluster have been also identified in Nosema grylli. PTP1 from N. locustae and N. grylli present a strong variability in the proline‐rich internal repeats, whilst PTP2s are highly conserved. It would be interesting to show whether this gene synteny conservation is related to a specific interaction between PTP1 and PTP2.
Tetrahymena cells and the quantitative two‐phase assay: visual assessment of chemokinesis and gravikinesis
Authors: OLESEN J. D. , HELLUNG‐LARSEN P. , LEICK V.
Published: April 2005

A two‐phase assay was used to assess chemokinesis and gravikinesis of Tetrahymena cells. METHOD: Tetrahymena thermophila cells were cultured without stirring at 36°C in a medium containing proteose peptone, yeast extract, glucose, and MOPS. Growing cells or cells in the stationary phase (1 million cells/ml) were used. Before assay, the cells were diluted 1+1. A semi‐microcuvette contained a 1‐ml bottom‐phase with NycoPrep™ Universal (5% w/v) and MOPS. Carefully, a 1‐mL top‐phase in the same buffer was layered on top of the bottom‐phase. Cells were either in the top‐ or the bottom‐phase. A commercially fed supplement for animals was used as chemoattractant in the phase opposite to the cells (Bioprotein, BP; Norferm, Norway). Changes in cell concentrations were monitored as optical density at 600 nm.RESULTS: (1) cells in top‐phase: −BP, 0.0001/30 min;+BP, 0.12/4 min. (2) cells in bottom‐phase: top‐phase exposed to a stream of nitrogen gas: −BP, 0.07/2 min; +BP 0.05/2 min; top‐phase not exposed to a stream of nitrogen gas: −BP, 0.08/2 min; +BP, 0.04/2 min. CONCLUSIONS: The BP proved to be an effective chemoattractant. Gravikinesis was observed with and without the presence of BP. Aerotaxis was not observed. The two‐phase assay is a sensitive, reliable, and quick procedure for the assessment of chemokinesis and gravikinesis.
Secondary endosymbioses and evolution of unicellular eukaryotes
Authors: OBORNÍK M. , GREEN B. R.
Published: April 2005

Many lines of evidence support the idea that the first chloroplast was the result of an endosymbiotic relationship between a cyanobacterium and a non‐photosynthetic eukaryote. Most of the cyanobacterial genes were lost, but a few remained in the chloroplast genome and as many as a thousand were transferred to the host nucleus. Genes encoding functions required by the chloroplast had to acquire presequences to target the products to the chloroplast. The situation gets more complicated when we consider the algae with chlorophyll c. They are the product of secondary endosymbiosis, where a putative red algal ancestor was engulfed by another non‐photosynthetic eukaryote, which retained the red algal chloroplast but eventually got rid of the rest of the cell. This left the chloroplast surrounded by two additional membranes: one derived from the red algal plasma membrane and the other from the host's phagocytic vacuole. In order for the endosymbiotic relationship to work, there must have been a substantial amount of gene transfer from the red algal nucleus to the host nucleus to support chloroplast functions. In the cryptophytes we even see an intermediate stage in this process, a relict nucleus (nucleomorph) in the periplastidal space between the outer two membranes and the original chloroplast envelope. Now that the draft genome sequence of the diatom Thalassiosira pseudonana (Diatom Genome Consortium) as well as genomes of rhodophyte Cyanidioschyzon merolae and green plants are available, it is possible to investigate the evolutionary history of plastid localized metabolic pathways. Phylogenetic analyses of nuclear‐encoded putatively plastid‐targeted enzymes showed that plastids obviously utilize enzymes not only of expected plastid (cyanobacterial) origin. Within the diatom, apicomplexan, plant and rhodophyte genomes, we have identified several enzymes that originate in α‐proteobacteria (mitochondria) or even in eukaryotic nucleus, but possess N‐terminal plastid‐targeting presequences. Although diatoms are, according to multiprotein phylogeny, related to Alveolates, some plastid‐related metabolic pathways show substantially different evolutionary pattern as well as, in silico, predicted localizations of involved enzymes.
Functional frataxin homologue in hydrogenosomes of Trichomonas vaginalis
Authors: DOLEZAL P. , DANCIS A. , EMBLEY M. , TACHEZY J.
Published: April 2005

The cellular structure of Trichomonas vaginalis substantially differs from other eukaryotes. Instead of mitochondria it possesses hydrogenosomes where key metabolic reactions are mediated by FeS proteins. The gene expression and the activity of FeS proteins is fully dependent on the iron availability. Although hydrogenosomes contain a high amount of iron, there is no information about hydrogenosomal iron homeostasis and FeS cluster formation. In mitochondria, frataxin plays an unclear role in iron metabolism. The deficiency of frataxin in human causes Friedreich's ataxia, which is associated with an increased level of mitochondrial iron. It has been suggested that frataxin is involved in various functions such as FeS cluster formation or iron storage. We cloned T. vaginalis frataxin homologue (TvFTX) coding for a protein of 121 amino acids. Although lacking transcription initiator element in 5′UTR region, the expression of tvftx was verified by a nuclear run‐on assay. Moreover, the gene upregulation in iron deficiency was shown. The N‐terminal part of TvFTX contains an extension similar to presequences targeting proteins to hydrogenosomes and mitochondria. To identify its cellular localization we transfected T. vaginalis with TvFTX fused to HA tag. The immunodetection of HA tag specifically labeled hydrogenosomes and the corresponding cellular fraction. The hydrogenosomal targeting sequence however does not support the translocation into yeast mitochondria. Thus for the complementation of yeast frataxin homologue (YFH1) a mitochondrial targeting sequence is required. When expressed in mitochondria of δYFH1 cells TvFTX complements the mutant growth defect and restores the activity of aconitase (iron‐sulfur protein). Together with recent characterization of T. vaginalis cystein desulfurase these data indicate the conserved role of hydrogenosomes and mitochondria in FeS cluster assembly.
RNA binding proteins MRP1 (gBP21) and MRP2 (gBP25) are essential for editing and stability of a subset of mitochondrial mRNAs in procyclic Trypanosoma brucei
Authors: VONDRUSKOVÁ E. , BURG J. , ZÍKOVÁ A. , ERNST N.L. , STUART K. , BENNE R. , LUKES J.
Published: April 2005

In trypanosomes, MRP1 and MRP2 (previously called gBPx and gBPy, in which x and y indicate the MW of these proteins in a particular species) are guide (g)RNA binding proteins that are part of a large heteromeric complex that may play a role in U‐insertion/deletion editing of mitochondrial mRNAs. In order to shed more light on the function of these proteins, we generated procyclic Trypanosoma brucei cell lines in which the levels of MRP 1 and/or MRP2 mRNA were downregulated by RNA interference (RNAi). Here we report that the RNAi‐mediated knockdown of MRP1 and/or MRP2 resulted in severe growth inhibition and loss of both proteins. This loss occurred even in cells in which only one of the MRPs was targeted by RNAi, indicating a mutual dependence for stability of these proteins. The elimination of the MRPs substantially reduced the levels of edited cytB and RPS12 mRNAs, but resulted in little or no reduction of edited cox2, cox3 and A6 mRNAs, as measured in poisoned primer extension analyses. Surprisingly, we found a five‐fold increase in ND7 mRNA editing in MRP1+2 double knockdown cells. In addition, the knockdowns also resulted in reduction in the amounts of mRNAs that do not undergo RNA editing (cox1, ND4 and ND5 mRNAs), but little change was observed for mitoribosomal 12S rRNA. Together, the results indicate that in procyclic T. brucei, MRP1 and MRP2 play a role in transcript‐specific editing and other RNA processing activities.
Effects of Xenobiotic Compounds on Cell Activities in Euplotes crassus
Authors: TRIELLI FRANCESCA , AMAROLI ANDREA , OGNIBENE MARZIA , FALUGI CARLA , DELMONTE CORRADO MARIA U.
Published: April 2005

It is now widely accepted that Protists are relevant bioassays to be exploited for the study of environmental modifications due to the presence of xenobiotic compounds. In this work, we evaluated the possibility of utilizing Euplotes crassus, an interstitial marine ciliate, for the pre‐chemical screening of environmental sites, such as estuarine and coastal sediments. With this aim, we tested the sensitivity of E. crassus to exposure to three classes of pollutants: an organophosphate neurotoxic drug, basudin, largely used for pest control in agricultural sites, a toxic heavy metal, mercury (HgCl2), and an aromatic polycyclic hydrocarbon, benzopyrene (BP). We found a dose‐dependent effect of these compounds on cell viability at concentrations ranging from 1/102 v/v to 1/107 v/v for basudin, from 5 μM to 0.1 μM for HgCl2, and from 50 μM to 1 μM for BP. In particular, 100% mortality was caused by a 1‐h exposure to 1/105 v/v basudin, or 2 μM HgCl2, or 25 μM BP, and by a 24‐h exposure to 1/106 v/v basudin, 0.5 μM HgCl2, or 5 μM BP. A significant decrease in the daily mean fission rate (P<0.001) was found after exposure to 1/107 v/v basudin, or 0.25 μM HgCl2, or 1 μM BP. Moreover, as it is well known that the inhibition of acetylcholinesterase (AChE) activity represents a specific biomarker for neurotoxic drugs, we first detected this enzyme activity in E. crassus, using cytochemical, spectrophotometric, and electrophoretic methods; then, AChE activity was characterized by its sensitivity to specific AChE inhibitors and to variations in pH and temperature. Like AChE present in higher organisms, the AChE activity detected in E. crassus was inhibited by exposure to basudin. Conversely, exposure to HgCl2, or PB did not inhibit AChE activity, but caused a significant reduction in lysosomal membrane stability.
Primary cultures of rat retina as a cell model for studying Toxoplasma gondii interconversion
Authors: ANGELICI MARIA C. , CELANI SERENA , MALCHIODI‐ALBEDI FIORELLA , MATTEUCCI ANDREA
Published: April 2005

Toxoplasma gondii infection is an important cause of chorioretinitis, that may lead to blindness and glaucoma. The particular tropism of the parasite for retinal tissue suggests that retinal cell cultures, reproducing retinal microenvironment, may represent a suitable cell model for obtaining intracellular replication of the parasite and its stage conversion from tachyzoites to bradyzoites. To verify this hypothesis, we set up primary cultures of embryonic retina, obtained from Wistar rats at the 18th gestational day. Eyes were removed under sterile conditions and the retina was isolated under a stereomicroscope. After dissociation in trypsin, cells were seeded at high density (1−2 × 106 cells) onto poly‐l‐lysine‐coated 24‐well cell culture plates and grown in MEM with 10% foetal calf serum. Cultures were maintained in a humidified, 37°C incubator with 5% CO2. This culturing procedure yields a mixed cell population, where all the principal cell types of the retina are present, including neuronal cells (amacrine cells, photoreceptors, ganglionar cells), macroglial cells (Muller cells, astrocytes) and microglia. One week after T. gondii ME49 strain inoculation, cyst‐like structures were observed, firstly, enclosed in grape‐like clusters of round cells, then free in the medium. In these cysts, bradizoites were still infective up to 2 months, and they developed to the tachyzoite stage when re‐inoculated in a fibroblast monolayer. These results suggest that the microenvironment of retinal cell cultures may favour parasite interconversion from tachyzoite to bradyzoite. Studies are in course to monitor cell‐signallings (INF‐γ, TNF‐α and nitric oxide) that accompanies this phenomenon. This in vitro model may be a valuable tool to study the role of inflammatory process in the pathogenesis of ocular toxoplasmic infection and to test new drugs to prevent ocular toxoplasmosis.
Tetrahymena thermophila as whole cell biosensor for environmental biomonitoring
Authors: BARCHETTA SABRINA , LA TERZA ANTONIETTA , BUONANNO FEDERICO , BALLARINI PATRIZIA , MICELI CRISTINA
Published: April 2005

Whole‐cell‐based biosensors represent an innovative and efficient class of tools useful for environmental monitoring. The majority of these biosensors that have been developed up to now are mainly based on bacteria. However, bacteria sense toxicity in a different way from eukaryotes, thus limiting the extrapolation of results to higher eukaryotes and humans. So far, the most used eukaryotic sensing element is represented by yeast, which is generally viewed as the benchmark eukaryotic microorganism. We propose, as a valid alternative to the yeast‐based sensors, the use of genetically engineered cell lines of the ubiquitous freshwater ciliated protozoa Tetrahymena thermophila. Ciliates, and in particular Tetrahymena species, offer a number of suitable characteristics to be used as environmental sensors: (a) they occupy the first trophic levels and consequently are early warning indicators of cellular suffering; (b) they are available for most of the newly developed molecular genetic techniques; (c) they can be easily cultured and maintained in small volumes; and (d) their cultures can be frozen and maintained in liquid nitrogen. Moreover, the analysis of the recently sequenced macronuclear genome of Tetrahymena thermophila has revealed that this ciliate shares a degree of sequence conservation with human genes higher than that showed by other single‐celled eukaryotic model organisms, yeast included. These considerations make Tetrahymena an appealing bio‐system for toxicity assessment since it can provide information of direct relevance to human health. In this study, Tetrahymena thermophila cells were transfected with the circular vector pD5H8 containing the coding sequence of the reporter gene “green fluorescent protein” (GFP) under the control of the homologous hsp70 stress promoter, to generate a fluorescent‐inducible bioreporter strain able to detect general toxicity. Several classes of chemicals (inorganic and organic), physical stressors and also real environmental samples from contaminated sites were assayed using the bioreporter strain. The sensitivity of this assay was significantly higher than that measured by lethality tests using the same organism.
Effects of climacostol on normal and tumoral mammalian cell lines
Authors: BUONANNO FEDERICO , BRAMUCCI MASSIMO , IIO HIDEO , ORTENZI CLAUDIO
Published: April 2005

Climacostol, 1,3‐dihydroxy‐5‐[(Z)‐2′‐nonenyl]benzene, is a natural toxin contained in the extrusomal cortical granules of the heterotrich ciliate Climacostomum virens. It is used for chemical defence against predators such as the raptorial ciliate Dileptus margaritifer and its cytotoxic activity has been assessed on several species of ciliates such as Didinium nasutum, Paramecium caudatum, and Blepharisma japonicum (Miyake et al. 2003, Europ. J. Protistol., 39:25–36). On the basis of its chemical structure, climacostol may be classified into the large group of natural compounds known as resorcinolic lipids, that show antimicrobial, antiparasitic, and antitumoral activities (Kozubek et al. 2003, Cell Moll. Biol. Lett., 6:351–355). To explore the possibility to use climacostol in medical applications, we examined the effects of chemically synthesized climacostol (Masaki et al. 2004, Tetrahedron, 60:7041–7048) on the growth and proliferation of tumoral and normal mammalian cell lines: (1) human promyelocytic leukaemia cells, HL60; (2) human squamous carcinoma cells, A431; and (3) non‐tumoral cells derived from mice Leydig cells, TM3. It was observed that (1) a concentration of 10 μg/ml of climacostol exerts a strong cytotoxic activity on all cell lines used; (2) at lower concentrations of 10 ng/ml and 1 ng/ml, the effect of climacostol is limited to the inhibition of the cell growth; and (3) the normal TM3 cells are more resistant to climacostol than the two tumoral HL60 and A431cell lines. The dose‐dependent cytotoxic effects of climacostol encourage further investigation on the potential use of this ciliate toxin as an anti‐cancer chemical.
Unusual tubulins with deviant genetic code in the hypotrich ciliate Euplotidium itoi
Authors: HARTMANN MANUELA , LUDWIG WOLFGANG , SCHLEIFER KARL‐HEINZ , PETRONI GIULIO
Published: April 2005

We characterized nine macronuclear tubulin gene minichromosomes in the hypotrich ciliate Euplotidium itoi: two α‐tubulin genes, five β‐tubulin genes, and two γ‐tubulin genes. In particular, one peculiar β‐tubulin gene was identified. It shows only 81.5% amino acid similarity to the other characterized β‐tubulin types and an UAG stop codon in frame. Based on this result we started to analyse stop codon usage in Euplotidium itoi macronuclear tubulin genes and some unrelated macronuclear minichromosomes. Most of the ciliates independently evolved diverse non‐canonical genetic codes. Differences to the standard code consist in the reassignment of one or two of the three standard stop codons to encode a particular amino acid. Class Spirotrichea represents a particular case comprising two groups of organisms that do not share any stop codon. In oxytrichids, UAR is translated as glutamine and UGA is the single stop codon. This is in contrast to the genus Euplotes, close relatives of Euplotidium, in which UAR encodes a stop signal and UGA is translated as cysteine. Fourteen complete genes were characterized in Euplotidium itoi; in all cases UAA revealed as the single triplet used to code a stop, UGA was never and UAG rarely observed in frame. The UAG stop codon was observed in the peculiar type 3 β‐tubulin gene, in two dynein partial sequences and in some other putative genes. We could confirm the active expression of three of these genes, the unusual β‐tubulin, a putative lipase and a possible steroid‐binding protein, by RT‐PCR. Finally, a detailed analysis on the structure of the macronuclear tubulin genes, including nucleotide and amino acid differences among tubulin gene paralogs, polyadenylation sites, putative chromosome fragmentation sites, ATGC‐content in coding and non‐coding regions, telomere sequence and length will be presented.
Protistan Diversity Estimates Based on 18S rDNA from Seawater Incubations in the Western North Atlantic1
Authors: COUNTWAY PETER D. , GAST REBECCA J. , SAVAI PRATIK , CARON DAVID A.
Published: April 2005

Abstract. Cloning/sequencing and fragment analysis of ribosomal RNA genes (rDNA) are becoming increasingly common methods for the identification of microbial taxa. Sequences of these genes provide many additional taxonomic characters for species that otherwise have few distinctive morphological features, or that require involved microscopy or laboratory culture and testing. These same approaches are now being applied with great success in ecological studies of natural communities of microorganisms. Extensive information on the composition of natural microbial assemblages is being amassed at a rapid pace through genetic analyses of environmental samples and comparison of the resulting genetic information with well‐established (and rapidly growing) public databases. We examined microbial eukaryote diversity in a natural seawater sample from the coastal western North Atlantic Ocean using two molecular biological approaches: the cloning and sequencing of rRNA genes and by fragment analysis of these genes using the terminal restriction fragment length polymorphism (T‐RFLP) method. A simple experiment was carried out to examine changes in the overall eukaryote (largely protistan) diversity and species composition (phylotype diversity) of a natural microbial assemblage when a seawater sample is placed in a container and incubated at ambient light and temperature for 72 h. Containment of the natural seawater sample resulted in relatively minor changes in the overall eukaryote diversity (species richness) obtained by either molecular method at three time points (time‐zero, time‐24 h, time‐72 h). However, substantial changes in the dominance of particular eukaryote phylotypes took place between the three sampling times. Only 18% of the total number of phylotypes observed in the study were observed at all three time points, while 65% (108 of 165) phylotypes were observed only at a single time point (54 unique phylotypes initially, 37 more unique phylotypes at 24 h, and 17 more at 72 h). The results of this study indicate that a high diversity of protistan taxa existed in the original seawater sample at very low abundance, and thus were not observed in the initial characterization of community structure. Containment resulted in significant shifts in the dominance of these taxa, enabling the presence of previously unobserved phylotypes to be documented after 24 or 72 h of incubation.
Detection of the Dinozoans Pfiesteria piscicida and P. shumwayae: A Review of Detection Methods and Geographic Distribution1
Authors: RUBLEE PARKE A. , REMINGTON DAVID L. , SCHAEFER ERIC F. , MARSHALL MICHAEL M.
Published: April 2005

Abstract. Molecular methods, including conventional PCR, real‐time PCR, denaturing gradient gel electrophoresis, fluorescent fragment detection PCR, and fluorescent in situ hybridization, have all been developed for use in identifying and studying the distribution of the toxic dinoflagellates Pfiesteria piscicida and P. shumwayae. Application of the methods has demonstrated a worldwide distribution of both species and provided insight into their environmental tolerance range and temporal changes in distribution. Genetic variability among geographic locations generally appears low in rDNA genes, and detection of the organisms in ballast water is consistent with rapid dispersal or high gene flow among populations, but additional sequence data are needed to verify this hypothesis. The rapid development and application of these tools serves as a model for study of other microbial taxa and provides a basis for future development of tools that can simultaneously detect multiple targets.
Periodontal disease awareness among pregnant women and its relationship with socio‐demographic variables
Authors: Alwaeli HA , Al‐Jundi SH
Published: April 2005

Certain conditions may have an affect on gingival status and may aggravate pre‐existing disease, especially in persons with poor oral hygiene. Pregnancy is one of these conditions. In addition, there is evidence in the literature suggesting an association between periodontal diseases in pregnant women and giving birth to premature, low‐birth infants. The purpose of this study was to evaluate the degree of periodontal health knowledge, and awareness, among pregnant women in Jordan. This was carried out by cross‐sectional survey using self‐administered, structured questionnaires distributed at six maternity care centres in Irbid City. Questionnaire items addressed personal and socio‐demographic variables and periodontal health awareness and knowledge of pregnant women. Women (n = 300) were randomly chosen from attendants to these centres. Of the 300 questionnaires distributed, only 275 pregnant women ages 16–45 years with a mean of 29 year responded and were included in the study. Data were analysed by chi‐squared test with the level of significance set at (P < 0.05). About one‐third of the participants had secondary level of education; it appeared that a minority of the pregnant women had knowledge or ability to identify dental plaque (16.4%) and its harmful effects (22.5%), while most of them (88%) were aware that bleeding gums indicated the presence of periodontal disease. The differences in the responses to knowledge questions were only significant for question number one (What is plaque?) among different educational levels and groups. This study revealed that (71.6%) of the pregnant women knew the main cause of gum disease; however 56% of them do not believe that frequency of teeth brushing should be increased during pregnancy and only 5.1% believed there might be a relationship between gum diseases and premature labour. Knowledge and awareness for pregnant women about their teeth and gingival condition is generally poor. Pregnant women need accurate information about their teeth and oral health. Simple educational preventive programmes on oral self‐care and disease prevention before and during pregnancy should be provided to improve oral health.
The use of the Internet to access oral health‐related information by patients attending dental hygiene clinics
Authors: Harris CE , Chestnutt IG
Published: April 2005

Background:  The degree to which patients access the Internet for oral health‐related information is unknown.

Objectives:  This study was designed to determine if patients attending student dental hygiene clinics had: (1) accessed the Internet, (2) used the Internet as a source of information on both general health and oral health, (3) bought oral health care products on‐line, and (4) were interested in being directed to appropriate on‐line oral health resources by dental professionals.

Methods:  A self‐complete questionnaire was administered to a quota sample of 280 new and returning patients attending the student dental hygiene clinic at either the Cardiff Dental Hospital or a nearby Primary Dental Care Unit.

Results:  A total of 269 (96%) patients consented to participate in the study. Of these, 147 (54.6%) had accessed the Internet, the majority (61.2%) from home. Internet use was significantly related to both patient age and level of education (P < 0.001). In total, 59 patients claimed accessing the Internet for information on general health topics. Only eight respondents had used the Internet for information on oral health/dental topics, with seven reporting that they found the information useful. On‐line purchase of oral health products was reported by seven patients. However, 117 (43.5% of all respondents) patients indicated that they would be interested in the Internet as a future source of oral health information and 112 (41.6%) would appreciate being directed to appropriate sites by their dentist or dental hygienist.

Conclusions:  Patients attending student dental hygiene clinics make little use of the Internet for oral health information. However, reported interest in future access is such that dental professionals should develop and harness the potential of the Internet as an educational resource.
The Global CAPM and a Firm's Cost of Capital in Different Currencies
Authors: O'Brien Thomas J.
Published: April 2005

In an integrated global market, a firm's cost of capital expressed in one currency should be consistent with its cost of capital expressed in another currency. This article presents and illustrates a process for estimating consistent costs of capital in different currencies for a U.K. based multinational. In so doing, it uses a simple, easy‐to‐use version of the global CAPM that attempts to incorporate the effect of uncertain exchange rates by calculating exchange rate “betas.” As argued in the previous article, at least part of a company's currency exposure is systematically related to the global market and thus should be treated as a component of the firm's systematic equity risk.

For example, the U.K. firm featured in this article is shown to have an exchange rate beta of 0.20 from the perspective of a U.S. investor. This implies that a 10% return on the global market in U.S. dollars tends to be associated with a 2% change in the U.S. dollar value of the British pound. One interesting consequence of incorporating exchange risk in this fashion is that two firms with identical U.S. global betas and costs of equity will have different expected returns expressed in another currency if they have different exposures to that currency.
BANKING REFORM IN ASIA: A PROGRESS REPORT
Authors: Freris Andrew F.
Published: April 2005

This article surveys and quantifies the progress made by five Asian economies during the period 1997–1999 in the reform and restructuring of their banking systems, paying particular attention to the very different circumstances and policy solutions in each of them. It develops a simple and objective scoring system of the progress achieved—one that puts South Korea at the top, Japan and Malaysia in the middle, and Thailand and Indonesia at the bottom. The relative success achieved in South Korea and Malaysia is attributed in part to their establishment of “Asset Management Corporations,” bodies charged with buying non‐performing loans, managing (and in some cases restructuring) the assets underlying the loans, and eventually reselling the loans. This practice has allowed for a more direct approach to corporate restructuring.
THE ASIAN CRISIS: LESSONS FOR CRISIS MANAGEMENT AND PREVENTION
Authors: Brealey Richard
Published: April 2005

The succession of financial crises that swept through Asia, Russia, and Latin America in 1997 and 1998 has led to considerable debate about both the role of the IMF and possible actions to limit further crises. Some have argued that international financial markets do not function well and are subject to periodic contagious panics that can be stemmed by an international lender of last resort. This paper argues that the IMF has neither the resources nor the superior information to play such a role. However, there may be a need for an international financial institution that can use its leverage to secure policy changes in the affected countries.

The paper also takes issue with the view that the IMF is simply bailing out imprudent international banks and that measures are needed to “bail them back in” again. The source of the Asian crisis lay in the real economy, but the effect of the crisis was greatly exacerbated by the financial structure of the affected countries. In particular, much of the risk was borne by domestic banks that borrowed dollars in the short‐term interbank market and made longerterm loans in the local currency. Public policy needs to be directed to ensuring that the risks in emerging markets are distributed efficiently across both foreign and domestic investors. That involves greater use of equity finance and structured debt, as well as possibly a more direct role for foreign banks in funding local activities.
CONVERTIBLE BONDS: MATCHING FINANCIAL AND REAL OPTIONS
Authors: Mayers David
Published: April 2005

For companies whose value consists in large part of “real options”‐ growth opportunities that may (or may not) materialize‐convertible bonds may offer the ideal financing vehicle because of the matching financial options built into the securities. This paper proposes that convertible debt can be a key element in a financing strategy that aims not only to fund current activities, but to give companies access to low‐cost capital if and when their real investment options turn out to be valuable. In this sense, convertibles can be seen as the most cost‐effective solution to a sequential financing problem‐how to fund not only today's activities, but also tomorrow's growth opportunities (some of them not yet even foreseeable).

For companies with real options, the ability of convertibles to match capital inflows with corporate outlays adds value by minimizing two sets of costs: those associated with having too much (particularly equity) capital (known as “agency costs of free cash flow”) and those associated with having too little (“new issue” costs). The key to the cost‐effectiveness of convertibles in funding real options is the call provision. Provided the stock price is “in the money” (and the call protection period is over), the call gives managers the option to force conversion of the bonds into equity. If and when the company's investment opportunity materializes, exercise of the call feature gives the firm an infusion of new equity (while eliminating the debt service burden associated with the convertible) that enables it to carry out its new investment plan.

Consistent with this argument, the author's recent study of the investment and financing activities of 289 companies around the time of convertible calls reports significant increases in capital expenditures starting in the year of the call and extending three years after. The companies also showed increased financing activity following the call, mainly new long‐term debt issues (many of them also convertibles) in the year of the call.
INNOVATIONS IN PARTITIONING A SHARE OF STOCK
Authors: Francis Jack Clark , Bali Rakesh
Published: April 2005

This paper presents a synthesis of innovations in the slicing and dicing of cash flows of a share of a firm's common stock. It begins by discussing PRIMEs and SCOREs, then Unbundled Stock Units (USUs), and finally three proposed hybrid equity options called DIVS, OWLS, and RISKS.

Decomposing a share of stock into components that can be traded separately allows investors to choose between the different investment attributes constituting the underlying share. An investor who desires only cash dividend income may buy only DIVS, and another who values capital appreciation but not current income can purchase only the residual claim.

Derivatives seem to go through a developmental process that is analogous to the biological phenomenon of natural selection and adaptation. The engines that drive this evolutionary process are changing domestic and international market conditions, international tax and regulatory arbitrage, and, of course, the financial innovators who learn from their own mistakes, and from the experience of others. These innovators continually develop new products that represent improvements over the old, if only by their ability to adhere more closely to the guidelines laid down by the regulators. The histories of PRIMEs and SCOREs, of USUs, and, most recently, of DIVS, OWLS and RISKS together provide a nice illustration of this developmental process.
THE HISTORY OF FINANCE: AN EYEWITNESS ACCOUNT
Authors: Miller Merton H.
Published: April 2005

In this account of the evolution of finance theory, the “father of modern finance” uses the series of Nobel Prizes awarded finance scholars in the 1990s as the organizing principle for a discus‐sion of the major developments of the past 50 years. Starting with Harry Markowitz's 1952 Journal of Finance paper on “Portfolio Selection,” which provided the mean‐variance frame‐work that underlies modern portfolio theory (and for which Markowitz re‐ceived the Nobel Prize in 1990), the paper moves on to consider the Capi‐tal Asset Pricing Model, efficient mar‐ket theory, and the M & M irrelevance propositions. In describing these ad‐vances, Miller's major emphasis falls on the “tension” between the two main streams in finance scholarship: (1) the Business School (or “micro normative”) approach, which focuses on investors ‘attempts to maximize returns and cor‐porate managers’ efforts to maximize shareholder value, while taking the prices of securities in the market as given; and (2) the Economics Depart‐ment (or “macro normative”) approach, which assumes a “world of micro optimizers” and deduces from that assumption how the market prices actually evolve.

The tension between the two ap‐proaches is resolved, and the two streams converge, in the final episode of Miller's history–the breakthrough in option pricing accomplished by Fischer Black, Myron Scholes, and Rob‐ert Merton in the early 1970s (for which Merton and Scholes were awarded the Nobel Prize in 1998, “with the late Fischer Black everywhere ac‐knowledged as the third pivotal fig‐ure”). As Miller says, the Black‐Scholes option pricing model and its many successors “mean that, for the first time in its close to 50‐year history, the field of finance can be built, or…rebuilt, on the basis of ‘observable’ magnitudes.” That option values can be calculated (almost entirely) with observable vari‐ables has made possible the spectacu‐lar growth in financial engineering, a highly lucrative activity where the prac‐tice of finance has come closest to attaining the precision of a hard sci‐ence. Option pricing has also helped give rise to a relatively new field called “real options” that promises to revolu‐tionize corporate strategy and capital budgeting.

But if the practical applications of option pricing are impressive, the op‐portunities for further extensions of the theory by the “macro normative” wing of the profession are “vast,” in‐cluding the prospect of viewing all securities as options. Thus, it comes as no surprise that when Miller asks in closing, “What would I specialize in if I were starting over and entering the field today?,” the answer is: “At the risk of sounding like the character in ‘The Graduate,’ I reduce my advice to a single word: options.”
STRATEGY AND SHAREHOLDER VALUE CREATION: THE REAL OPTIONS FRONTIER
Authors: Amram Martha , Kulatilaka Nalin
Published: April 2005

The current interest in real options reflects the dramatic increase in the uncertainty of the business environment. Viewed narrowly, the real options approach is the extension of financial option pricing models to the valuation of options on real (that is, nonfinancial) assets. More broadly, the real options approach is a way of thinking that helps managers formulate their strategic options—the future opportunities that are created by today's investments—while considering their likely effect on shareholder value.

But if the real options framework promises to link strategy more closely to shareholder value creation, there are some major challenges on the frontier of application. In the first part of this paper, the authors tackle the question, “What is really new about real options, and how does the approach differ from other wellestablished ways to make strategic decisions under uncertainty?” This article provides a specific definition of real options that relies on the ability to track marketpriced risk. Using examples from oil exploration and pharmaceutical drug development, the authors also show how specific features of the industry and the application itself determine the usefulness of the real options approach.

The second part of the paper addresses the question: Given the many differences between real and financial options, how should a real options application be framed? The authors examine the use of real options in the valuation of Internet companies to demonstrate the required judgment and tradeoffs in the framing of real options applications. The case of Webvan, an online grocer, is used to illustrate the inter‐action between strategy, execution, and valuation.
AN INTRODUCTION TO GAME THEORY AND BUSINESS STRATEGY
Authors: Brickley James , Smith Clifford , Zimmerman Jerold
Published: April 2005

Sound managerial decision making often requires “putting yourself behind your rivals' desk.” Assuming rivals are rational and acting in their selfinterest, what decisions are they likely to make and how are they likely to respond to your actions? A complicating factor is that rivals' optimal choices typically will depend on their expectations of what you will do; their expectations in turn depend on their assessments of your expectations about them. This type of circularity or recursive thinking might appear to make the overall problem completely intractable. Yet, this situation is precisely where game theory is most useful.

This article introduces the basic elements of game theory within the context of business strategy and shows how managers might use these tools in decision making. This analysis also provides managers with a richer understanding of competition within different market settings. For example, it provides insights into why there is fierce competition in some concentrated industries (such as commercial aircraft), but not in others. Although the authors focus primarily on interactions among rival firms in product markets, these concepts also are useful to managers when dealing with other parties, such as suppliers, employees, or gov‐ernment officials.