The First Record of the Invasive Asian Fish Tapeworm (Schyzocotyle Acheilognathi) from An Endemic Cichlid Fish in Madagascar.

The Asian fish tapeworm, Schyzocotyle acheilognathi (Yamaguti, 1934) (Cestoda: Bothriocephalidea), is an invasive parasite of freshwater fishes that have been reported from more than 200 freshwater fish worldwide. It was originally described from a small cyprinid, Acheilognathus rombeus, in Japan but then has spread, usually with carp, minnows or guppies, to all continents including isolated islands such as Hawaii, Puerto Rico, Cuba or Sri Lanka. In the present account, we report the first case of the infection of a native cichlid fish, Ptychochromis cf. inornatus (Perciformes: Cichlidae), endemic to Madagascar, with S. acheilognathi. The way of introduction of this parasite to the island, which is one of the world's biodiversity hotspots, is briefly discussed.

Novel insights into the electrochemical detection of nitric oxide in biological systems.

In recent years, microsensor technologies have made a rapid expansion into different fields of physical sciences, engineering, and biomedicine. For analyses of various biomolecules, novel sensors and detection platforms in the electrochemical field have been reported recently. The most important applications based on microelectromechanical systems dramatically reduce the need of manipulation steps with samples, while improving data quality and quantitative capabilities. This is also the case of a special class of electrochemical sensors that allow direct, real-time and non-invasive measurements of nitric oxide, whose determination is crucial for the purposes of basic research, as well as of preclinical and clinical studies. Therefore, this minireview will focus on the description of recent discoveries in the electrochemical determination of nitric oxide, released in different in vitro systems.

Eukaryotic DNA primase.

Eukaryotic DNA primase initiates the synthesis of all new DNA strands by synthesizing short RNA oligomers on single-stranded DNA. Additionally, primase helps couple replication and repair and is critical for telomere maintenance and, therefore, chromosome stability. In light of the many aspects of DNA metabolism in which primase is involved, understanding the unique features of the mechanism of this enzyme and how it interacts with other proteins will greatly advance our knowledge of DNA replication and repair.

Suppression of the tapeworm order Pseudophyllidea (Platyhelminthes: Eucestoda) and the proposal of two new orders, Bothriocephalidea and Diphyllobothriidea.

Pseudophyllidea van Beneden in Carus, 1863, a well recognised order of tapeworms (Platyhelminthes: Eucestoda), is suppressed because it is composed of two phylogenetically unrelated groups, for which the new names Bothriocephalidea and Diphyllobothriidea are proposed. The new orders differ from each other in the following characters: (i) position of the genital pore: on the dorsal, dorso-lateral or lateral aspects and posterior to the ventral uterine pore in the Bothriocephalidea versus on the ventral aspect of segments and anterior to the uterine pore in the Diphyllobothriidea; (ii) the presence of a muscular external seminal vesicle in the Diphyllobothriidea, which is absent in the Bothriocephalidea; (iii) the presence of a uterine sac in the Bothriocephalidea, which is absent in the Diphyllobothriidea; and (iv) the spectrum of definitive hosts: mainly teleost fishes, never homoiothermic vertebrates in the Bothriocephalidea, versus tetrapods, most frequently mammals, in the Diphyllobothriidea, with species of Diphyllobothrium, Spirometra and Diplogonoporus parasitic in humans. The Diphyllobothriidea, which includes 17 genera in four families (Digramma is synonymised with Ligula), is associated with cestode groups that have a range of plesiomorphic characters (Haplobothriidea and Caryophyllidea), whereas the Bothriocephalidea, consisting of 41 genera grouped in four families, is the sister-group to the 'acetabulate' or 'tetrafossate' cestodes, which are generally regarded as having derived characters.

Diversity and distribution of fish tapeworms of the "Bothriocephalidea" (Eucestoda).

Recent studies have demonstrated the invalidity of the Pseudophyllidea, a long-term recognised order of tapeworms (Platyhelminthes: Cestoda), typical in possessing two dorsoventrally situated attachment organs called bothria. In fact, cestodes parasitic in tetrapods, especially mammals including man, form a relatively basal group called provisionally the "Diphyllobothriidea", whereas tapeworms occurring in freshwater and marine fish, with a few taxa known from amphibians (frogs and newts), belong to a more derived clade, for which the name "Bothriocephalidea" is tentatively proposed. Revision of the "Bothriocephalidea", based on literary data, study of type- and voucher specimens and extensive newly collected material made it possible to critically review the species composition of the group and to prepare a tentative list of its valid species. Out of 305 nominal taxa, only 125 species are considered to be valid. In addition, the spectrum of definitive hosts and geographical distribution of bothriocephalideans are briefly discussed.

Paraphyly of the Pseudophyllidea (Platyhelminthes: Cestoda): circumscription of monophyletic clades based on phylogenetic analysis of ribosomal RNA.

Phylogenetic relationships of cestodes of the order Pseudophyllidea (Platyhelminthes: Cestoda) were examined using sequences of complete small subunit and partial (D1-D3 region) large subunit nuclear rDNA of members of all pseudophyllidean families. The results provide evidence of paraphyly of the order as indicated by previous molecular phylogenetic analyses based on a much lower number of species sequenced. Pseudophyllidean tapeworms represent an artificial assemblage comprising two unrelated clades. "Bothriocephalidea" is formed by four families sensu Bray et al. (1994), namely Bothriocephalidae, Echinophallidae, Philobythiidae and Triaenophoridae, whereas two other families, Diphyllobothriidae and Cephalochlamydidae, give rise to the "Diphyllobothriidea". The present results indicate that "Bothriocephalidea" forms the most derived clade of all difossate and tetrafossate/bothriate tapeworm lineages which are considered to be basal relative to the rest of tetrafossate/bothridiate and acetabulate cestodes. By contrast, "Diphyllobothriidea", which includes medically important parasites (Diphyllobothrium and Spirometra), appeared more basal, without a clearly resolved position within other difossate tapeworm lineages.

Morphometric analysis of four species of Eubothrium (Cestoda: Pseudophyllidea) parasites of salmonid fish: an interspecific and intraspecific comparison.

Four species of the genus Eubothrium (E. crassum, E. fragile, E. rugosum and E. salvelini) were subjected to morphometric comparison. Discriminant analysis was conducted utilising 17 characters measured on the scolex and strobila of 101 specimens. Univariate statistics were first used to detect features that were useful for separating individual Eubothrium species and two different host populations of E. salvelini. Subsequent multivariate discriminant analysis, combining all the measured variables, made it possible to separate all four species. A comparison of the four taxa revealed that (1) E. fragile is the most distinct species, possessing a much smaller scolex than the other congeners, and its similarity with the other marine species E. crassum is not proven; (2) the two freshwater taxa, E. rugosum and E. salvelini are the most similar; (3) the characters most suitable for species differentiation are the length of the scolex, the width of the apical disc, the width of the neck and its area, the width of eggs and the number of testes; (4) the width of the apical disc was confirmed to be the most stable character at the intraspecific level (within E. salvelini host populations) and is therefore considered to be a trait of the highest discriminative power in the subset of four Eubothrium species.

Inhibition of UDP-N-acetylglucosamine import into Golgi membranes by nucleoside monophosphates.

The specificity of the UDP-N-acetylglucosamine (UDP-GlcNAc) translocator for the binding of nucleoside monophosphates (NMPs) and nucleotide-sugars was examined in order to develop a quantitative understanding of how this enzyme recognizes its substrates and to provide a framework for development of novel drugs that target glycosylation. Competition studies reveal that tight binding requires a complete ribose ring and a 5'-phosphate. The enzyme is extremely tolerant to changes at the 3'-position, and the presence of 3'-F actually increases binding of the NMP to the enzyme. At the 2'-position, substitutions in the ribo configuration are well tolerated, although these same substitutions greatly diminish binding when present in the ara configuration. For the base, size appears to be the key feature for discrimination. The enzyme tolerates changing the C-4 oxygen of uridine to an amino group as well as substituting groups containing one or two carbons at C-5. However, substitution of groups containing three carbons at C-5, or exchange of the pyrimidine for a purine, greatly weakens binding to the translocator. Comparison of various UDP-sugars reveals that the UDP-GlcNAc translocator has lower affinity for UDP-N-acetylgalactosamine and UDP-glucose than for its cognate substrate and therefore indicates that this translocator requires both proper stereochemistry at C-4 and an aminoacetyl group at C-2. The impact of these observations on the design of more powerful nucleoside-based inhibitors of nucleotide-sugar import is discussed.

Polymerization of 2'-fluoro- and 2'-O-methyl-dNTPs by human DNA polymerase alpha, polymerase gamma, and primase.

Studies were undertaken to assess the ability of human polymerase alpha (pol alpha) and polymerase gamma (pol gamma) to incorporate 2'-fluoro- and 2'-O-methyldeoxynucleotides into DNA. In vitro DNA synthesis systems were used to detect incorporation and determine K(m) and V(max) for 2'-FdATP, 2'-FdUTP, 2'-FdCTP, 2'-FdGTP, 2'-O-MedATP, 2'-O-MedCTP, 2'-O-MedGTP, 2'-O-MedUTP, dUTP, UTP, and FIAUTP, in addition to normal deoxynucleotides. Pol alpha incorporated all 2'-FdNTPs except 2'-FdATP, but not 2'-O-MedNTPs. Pol gamma incorporated all 2'-FdNTPs, but not 2'-O-MedNTPs. In general, 2'-fluorine substitution decreased V(max)/K(m) 2'-FdUTP. Because kinetics of insertion of pol alpha can be affected by the nature of the primer, we examined the ability of pol alpha to polymerize 2'-fluoro- and 2'-O-MedATP and dGTP when elongating a primer synthesized by DNA primase. Under these conditions, both 2'-FdATP and 2'-FdGTP were polymerized, but 2'-O-MedATP and 2'-O-MedGTP were not. Primase alone could not readily polymerize these analogs into RNA primers. Previous studies showed that 2'-deoxy-2'-fluorocytosine (2'-FdC) is incorporated by several non-human DNA polymerases. The current studies showed that human polymerases can polymerize numerous 2'-FdNTPs but cannot polymerize 2'-O-MedNTPs.

Two new species of Aporhynchus (Cestoda: Trypanorhyncha) from deep water lanternsharks (Squaliformes: Etmopteridae) in the Azores, Portugal.

New collections of cestodes from the spiral intestines of the lanternsharks Etmopterus spinax and Etmopterus pusillus off the island of Faial, in the Azores, Atlantic Ocean, have yielded 2 new species of trypanorhynchs belonging to Aporhynchus. Both species share the distinctive lack of all elements of the rhyncheal system that are characteristic of this genus. The identity of Aporhynchus norvegicus is clarified to allow it to be distinguished from A. menezesi n. sp., which also parasitizes E. spinax. This new species differs conspicuously from its congeners in that its mature and gravid proglottids are wider than long, rather than longer than wide, and also in its lack of spinitriches on the scolex. Aporhynchus pickeringae n. sp., the new species from E. pusillus , differs from all of its congeners except A. norvegicus in that it is a relatively delicate worm with relatively fewer testes. It also possesses fewer proglottids and a wider pedunculus scolecis than does A. norvegicus. Sections through the scolex of A. menezesi n. sp. support use of the term bothriate, rather than difossate, in reference to the scolex configuration of some trypanorhynchs. A key to the 4 species of Aporhynchus is provided.

A new genus and two new species of Aporhynchidae (Cestoda: Trypanorhyncha) from catsharks (Carcharhiniformes: Scyliorhinidae) off Taiwan.

New collections of cestodes from the spiral intestines of catsharks (Carcharhiniformes: Scyliorhinidae) off Taiwan have led to the discovery of a new genus and 2 new species of trypanorhynchs. These taxa are relatively unique among trypanorhynchs in their lack of all elements of the rhyncheal apparatus. The new genus, Nakayacestus n. gen., is considered to belong with Aporhynchus in the Aporhynchidae. In addition to lacking the rhyncheal apparatus, these 2 genera share circum-medullary vitelline follicles, post-ovarian testes, and complex terminal genitalia consisting of accessory, external, and internal seminal vesicles. The 2 genera differ conspicuously in spinithrix configuration; whereas both species of Nakayacestus n. gen. bear scolex spinitriches that are bifid, trifid, or pectinate, species of Aporhynchus either lack scolex spinitriches entirely or possess spathulate spinitriches. The configuration of the bothria of the 2 genera also differ conspicuously. Whereas the bothria of Aporhynchus are sessile and generally do not extend beyond the lateral margins of the cephalic peduncle, those of Nakayacestus bear only a tenuous connection with the scolex proper, being conspicuously free both anteriorly and posteriorly and extending conspicuously beyond the cephalic peduncle. Futhermore, the boundary between the scolex and the strobila of members of the new genus is clearly delineated, whereas this distinction is ill-defined in species of Aporhynchus. Nakayacestus takahashii n. sp., the type of the new genus, was collected from the Broadmouth catshark, Apristurus macrostomus, and differs from Nakayacestus tanyderus n. sp., collected from the Blacktip sawtail catshark, Galeus sauteri, in being shorter, bearing a longer pedunculus scolecis, an ovary that is more posterior in the proglottid, and fewer post-ovarian testes. Furthermore, the 2 new species differ conspicuously from one another in the configuration of their scolex spinitriches.

Mechanism of calf thymus DNA primase: slow initiation, rapid polymerization, and intelligent termination.

The mechanism by which calf thymus DNA primase synthesizes RNA primers was examined. Primase first binds a single-stranded DNA template (KD << 100 nM) and can then slide along the DNA in order to find a start for initiating primer synthesis. NTP binding appears ordered, such that the NTP which eventually becomes the second nucleotide of the primer binds the E.DNA complex first. The NTP that becomes the second nucleotide of the primer thereby influences where primase initiates. Primer synthesis is remarkably slow (0.0027 s-1 at 20 microM NTP). The rate-limiting step is after formation of the E.DNA.NTP.NTP complex and before or during dinucleotide synthesis. After synthesis of the dinucleotide, additional NTPs are rapidly polymerized. Primase products are 2-10 nucleotides long. If the enzyme fails to synthesize a primer at least 7 nucleotides long, it reinitiates rather than dissociating from the template. Once a primer at least 7 nucleotides long has been generated, however, subsequent primase activity is inhibited. This inhibition is due to the generation of a stable primer-template complex, which likely remains associated with pol alpha.primase. The role of primase is to synthesize primers that pol alpha can elongate. The ability of primase to distinguish between primers at least 7 nucleotides long and shorter products therefore likely reflects the fact that pol alpha only utilizes primers at least 7 nucleotides long.

Inhibition of DNA primase by 9-beta-D-arabinofuranosyladenosine triphosphate.

9-beta-D-Arabinofuranosyladenosine triphosphate (araATP) is a potent inhibitor of DNA primase. Primase readily incorporates araATP into primers, and primers containing araAMP are then elongated by DNA polymerase alpha (pol alpha) upon addition of dNTPs. AraATP did not inhibit utilization of primers under conditions where the ability of pol alpha to elongate primers was independent of the dATP concentration. The fraction of primers elongated by pol alpha was reduced by araATP only when elongation was dependent upon the dATP concentration. When the Ki for primase was measured in terms of the inhibition of the synthesis of primers that can be utilized by pol alpha, we obtained Ki = 2.7 microM (37 degrees C) and 2.0 microM (25 degrees C). Inhibition was competitive with ATP. Inhibition of pol alpha activity by araATP was measured under conditions where primase-catalyzed primer synthesis was required for the pol alpha activity. The decreased pol alpha activity was due to primase inhibition, and at constant dATP, araATP inhibition was competitive with ATP and gave Ki = 1.2 microM, similar to the Ki for primase alone. Increasing the dATP concentration had no effect on inhibition. In combination with previously reported in vivo data, we conclude that DNA primase is the primary in vivo target of the arabinofuranosyl nucleotides, not pol alpha.

Mechanism of DNA polymerase alpha inhibition by aphidicolin.

Synthetic oligonucleotides of defined sequence were used to examine the mechanism of calf thymus DNA polymerase alpha inhibition by aphidicolin. Aphidicolin competes with each of the four dNTPs for binding to a pol alpha-DNA binary complex and thus should not be viewed as a dCTP analogue. Kinetic evidence shows that inhibition proceeds through the formation of a pol alpha.DNA.aphidicolin ternary complex, while DNase I protection experiments provide direct physical evidence. When deoxyguanosine is the next base to be replicated, Ki = 0.2 microM. In contrast, the Ki is 10-fold higher when the other dNMPs are at this position. Formation of a pol alpha.DNA.aphidicolin ternary complex did not inhibit the primase activity of the pol alpha.primase complex. Neither the rate of primer synthesis nor the size distribution of primers 2-10 nucleotides long was changed. Elongation of the primase-synthesized primers by pol alpha was inhibited both by ternary complex formation using exogenously added DNA and by aphidicolin alone.

Lactate reduction in Clostridium propionicum. Purification and properties of lactyl-CoA dehydratase.

Clostridium propionicum converts lactate to propionate (Cardon, B.P., and Barker, H.A. (1947) Arch. Biochem. Biophys. 12, 165-171). We have obtained a soluble system that carries out this conversion as well as the hydration of acrylate to lactate and the reduction of acrylate to propionate. 3-Pentynyl-CoA inhibits reduction of acrylate and lactate to propionate, but not hydration of acrylate to lactate by cell extracts. The conversion probably involves CoA esters. When [beta-2H3] lactate is used as a substrate, the rate of propionate formation is reduced 1.8-fold, and the methyl group of the resulting propionate has lost 1.4 deuterium atoms. These results are consistent with the intermediate formation of acrylate (acrylyl-CoA) in the conversion of D-lactate to propionate. Two proteins, which we designate E I and E II, were purified to greater than 90% homogeneity. Together, they catalyze the hydration of acrylyl-CoA to lactyl-CoA. E I has an apparent molecular mass of 27,000 daltons and is rapidly and irreversibly inactivated by O2. E II consists of two subunits of molecular mass 41,000 and 48,000 daltons and contains equal amounts of riboflavin and flavin mononucleotide. Hydration of acrylyl-CoA to lactyl-CoA requires Mg2+ and catalytic quantities of ATP. GTP can replace ATP, but ADP and adenylyl imidodiphosphate cannot. We were unable to detect any stable intermediate during acrylyl-CoA hydration. Finally, we proposed a mechanism for this reaction.

Misincorporation of nucleotides by calf thymus DNA primase and elongation of primers containing multiple noncognate nucleotides by DNA polymerase alpha.

Misincorporation of nucleotides by calf thymus DNA primase was examined using synthetic DNA templates of defined sequence. Primase seldom misincorporated NTPs during initiation of a new primer (i.e. polymerization of two NTPs to generate the dinucleotide). Following dinucleotide formation, however, primase readily misincorporated NTPs. Although the rate of misincorporation varied according to both the identity of the mismatch and the template sequence, primase is by far the least accurate nucleotide-polymerizing enzyme known. In some cases primase discriminated against incorrect NTPs by less than a factor of 100. After primase incorporated a noncognate nucleotide into the primer, the next correct NTP was readily added. Remarkably, primase could also polymerize consecutive noncognate nucleotides and generate primers containing multiple mismatches. Generation of a correctly base-paired primer-template negatively regulated further primer synthesis; however, generation of a primer-template containing multiple mismatches did not. After primase synthesized a primer containing multiple mismatches, the primer was transferred to the polymerase alpha active site via an intramolecular mechanism. Importantly, polymerase alpha readily elongated this primer if dNTPs were present. These data are discussed with respect to the question of why primase is required for DNA replication.

Arg304 of human DNA primase is a key contributor to catalysis and NTP binding: primase and the family X polymerases share significant sequence homology.

Comparison of the amino acid sequences of eucaryotic DNA primase and the family X polymerases indicates that primase shares significant sequence homology with this family. With the use of DNA polymerase beta (pol beta) as a paradigm for family X polymerases, these homologies include both the catalytic core domain/subunit of each enzyme (31 kDa domain of pol beta and p49 subunit of primase) as well as the accessory domain/subunit (8 kDa domain of pol beta and p58 subunit of primase). To further explore these homologies as well as provide insights into the mechanism of primase, we generated three mutants (R304K, R304Q, and R304A) of the p49 subunit at an arginine that is highly conserved between primase and the eukaryotic family X polymerases. These mutations significantly decreased the rate of primer synthesis, due primarily to a decreased rate of initiation, and the extent of impairment correlated with the severity of the mutation (A > Q > K). R304 also contributes to efficient utilization of the NTP that will become the 5'-terminus of the new primer, and these effects are at least partially mediated through interactions with the phosphates of this NTP. The implications of these results with respect to the structure and biological role of primase, as well as its relationship to the family X polymerases, are discussed.

Human DNA primase: anion inhibition, manganese stimulation, and their effects on in vitro start-site selection.

We examined the effects of Mn(2+) on eukaryotic DNA primase both in the presence and absence of 5 mM Mg(2+). In the absence of Mg(2+), Mn(2+)-supported primase activity to a level 4-fold greater than that obtained with Mg(2+) alone, and adding low levels of Mn(2+) (100 microM) to assays containing 5 mM Mg(2+) greatly stimulated primase. Increased activity was primarily due to more efficient utilization of NTPs, as reflected in a lower K(M) for NTPs. Under conditions of saturating NTPs, Mn(2+) had minimal effects on both the rate of initiation (i.e., dinucleotide synthesis) and processivity. The effects of Mn(2+) involve multiple metal binding sites on primase and may involve both the catalytic p49 subunit as well as the p58 subunit. Physiological levels of salt can inhibit primase activity due to the presence of an anion binding site and low levels of Mn(2+) significantly decreased this salt sensitivity. The implications of these results with respect to the biological role of primase are discussed.