Baculovirus expression and bioactivity of a soluble 140 kDa extracellular cleavage fragment of L1 neural cell adhesion molecule

L1 neural cell adhesion molecule is the founding member of the L1 subfamily of the immunoglobulin superfamily and plays an important role in the overall development of both the central and peripheral nervous systems, making it an attractive candidate for promoting neural regeneration following injury. Currently, L1 used for experimental studies is primarily mammalian-derived; however, the insect cell expression system described here provides an alternative source of recombinant L1 with equivalent bioactivity. A 140 kDa L1 fragment based on a physiological plasmin cleavage site in the extracellular domain was cloned and expressed with a C-terminal 6x histidine tag. Recombinant insect cell-derived L1 was analyzed by Western blot using an antibody to human L1 to confirm immunogenicity and to optimize infection conditions for recombinant L1 production. The recombinant protein was secreted by insect cells, efficiently purified under non-denaturing conditions using dialysis followed by metal affinity chromatography, and analyzed by SDS-PAGE to produce a single band of the expected approximate 140 kDa size. The bioactivity of insect cell-derived L1 was compared to mammalian-derived L1-Fc and poly-L-lysine (PLL) using chick embryonic forebrain neurons. The results show comparable, robust neurite outgrowth at 24h on insect cell-derived L1 and mammalian-derived L1-Fc, with significantly longer neurites than those observed on PLL. Future studies will examine the immobilization of L1 to biomaterial surfaces in physiologically appropriate orientation via the C-terminal 6x histidine tag and will investigate their application in promoting axonal regeneration in the injured nervous system.     

The effect of ephedra and caffeine on maximal strength and power in resistance-trained athletes

Caffeine and ephedrine-related alkaloids recently have been removed from International Olympic Committee banned substances lists, whereas ephedrine itself is now permissible at urinary concentrations less than 10 mug.mL. The changes to the list may contribute to an increased use of caffeine and ephedra as ergogenic aids by athletes. Consequently, we sought to investigate the effects of ingesting caffeine (C) or a combination of ephedra and caffeine (C + E) on muscular strength and anaerobic power using a double-blind, crossover design. Forty-five minutes after ingesting a glucose placebo (P: 300 mg), C (300 mg) or C + E (300 mg + 60 mg), 9 resistance-trained male participants were tested for maximal strength by bench press [BP; 1 repetition maximum (1RM)] and latissimus dorsi pull down (LP; 1RM). Subjects also performed repeated repetitions at 80% of 1RM on both BP and LP until exhaustion. After this test, subjects underwent a 30-second Wingate test to determine peak anaerobic cycling power, mean power, and fatigue index. Although subjects reported increased alertness and enhanced mood after supplementation with caffeine and ephedra, there were no significant differences between any of the treatments in muscle strength, muscle endurance, or peak anaerobic power. Our results do not support the contention that supplementation with ephedra or caffeine will enhance either muscle strength or anaerobic exercise performance.     

Tormopsolus attenuatus n. sp. (Digenea: Acanthocolpidae) from Seriola hippos (Perciformes: Carangidae), Western Australia,with some observations on the relationships in the genus

A new acanthocolpid, Tormopsolus attenuatus n. sp., is described from the carangid Seriola hippos from waters off the coast of SW Australia. A cladistic analysis of the genus using 31 morphological and metrical characters indicates that the closest species is T. asiatica Parukhin, 1976 status amend. (previously T. orientalis asiatica). T. attenuatus differs from T. asiatica in its smaller overall dimensions, more attenuated outline, relatively shorter post-testicular region, vitelline interruption at the level of the ovary, papillae on the oral sucker and, possibly, much longer genital atrium. Other similar species, T. orientalis Yamaguti, 1934 and T. medius Reimer, 1983, differ in having interruptions of the vitelline fields at the level of both testes and at the level of the ovary.     

A redescription of Homalometron senegalense Fischthal & Thomas, 1972 (Digenea: Apocreadiidae) from Synaptura kleinii (Teleostei) of the western Mediterranean

The apocreadiid digenean Homalometron senegalense is redescribed from the soleid fish Synaptura kleinii from off Corsica in the western Mediterranean. For the first time, lymphatic vessels are described for this species, and the implications of this in the systematics of the Apocreadiidae discussed. This species is considered closest to H. galaicus and H. wrightae, both also reported from soleid hosts. The concept of Apocreadiidae espoused is that most recently developed by Cribb & Bray (1999).     

Faustulid trematodes (Digenea) from marine fishes of Australia

Twelve species of faustulid trematode are described or redescribed from Australian marine fishes. Bacciger lesteri Bray, 1982 and B. sprenti Bray, 1982 are redescribed from Selenotoca multifasciata from Moreton Bay. It is suggested that the original host record for these species, Mugil sp., was incorrect. The genera Discogastroides, Odontocotyle and Pseudodiscogasteroides are synonymised with Paradiscogaster. The new combinations Paradiscogaster arabi (Hafeezullah & Siddiqi, 1970), P. hainanensis (Shen, 1970), P. indicus (Srivastava, 1939), P. macrostomus (Shimazu & Kamegai, 1990), P. ostracii (Yamaguti, 1934) and P. pritchardae (Gupta & Ahmad, 1978) are proposed. Discogasteroides hawaiensis Hanson, 1955 is synonymised with P. ostracii. P. macrostomus and P. ostracii are redescribed from Ostracion meleagris and O. cubicus from the Great Barrier Reef. P. farooqii Hafeezullah & Siddiqi, 1970 is redescribed from Monodactylus argenteus from Moreton Bay. The following new species are described: P. machidai n. sp. from Pomacanthus semicirculatus and P. sexstriatus from the Great Barrier Reef, P. dweorg n. sp. from Meuschenia galii, P. lobomyzon n. sp. from Tilodon sexfasciatus and P. habilis n. sp. from Pelates octolineatus, all from Western Australia. Antorchis pomacanthi (Hafeezullah & Siddiqi, 1970) Machida, 1975 is redescribed from Pomacanthus semicirculatus and P. sexstriatus from the Great Barrier Reef. The new combination Antorchis intermedius (Madhavi, 1975) is proposed for Parantorchis intermedius. Parayamagutia ostracionis is redescribed from O. cubicus from the Great Barrier Reef. Trigonocryptus conus is redescribed from Arothron hispidus from South-east Queensland and from A. nigropunctatus from the Great Barrier Reef. The new combination Trigonocryptus australiensis (Kurochkin, 1970) is proposed for Pseudodiscogasteroides australiensis. The Echinobrevicecinae is reduced to synonymy with the Faustulidae.     

V4 region of small subunit rDNA indicates polyphyly of the Fellodistomidae (Digenea) which is supported by morphology and life-cycle data

There is no morphological synapomorphy for the disparate digeneans, the Fellodistomidae Nicoll, 1909. Although all known life-cycles of the group include bivalves as first intermediate hosts, there is no convincing morphological synapomorphy that can be used to unite the group. Sequences from the V4 region of small subunit (18S) rRNA genes were used to infer phylogenetic relationships among 13 species of Fellodistomidae from four subfamilies and eight species from seven other digenean families: Bivesiculidae; Brachylaimidae; Bucephalidae; Gorgoderidae; Gymnophallidae; Opecoelidae; and Zoogonidae. Outgroup comparison was made initially with an aspidogastrean. Various species from the other digenean families were used as outgroups in subsequent analyses. Three methods of analysis indicated polyphyly of the Fellodistomidae and at least two independent radiations of the subfamilies, such that they were more closely associated with other digeneans than to each other. The Tandanicolinae was monophyletic (100% bootstrap support) and was weakly associated with the Gymnophallidae (<50–55% bootstrap support). Monophyly of the Baccigerinae was supported with 78–87% bootstrap support, and monophyly of the Zoogonidae + Baccigerinae received 77–86% support. The remaining fellodistomid species, Fellodistomum fellis, F. agnotum and Coomera brayi (Fellodistominae) plus Proctoeces maculatus and Complexobursa sp. (Proctoecinae), formed a separate clade with 74–92% bootstrap support. On the basis of molecular, morphological and life-cycle evidence, the subfamilies Baccigerinae and Tandanicolinae are removed from the Fellodistomidae and promoted to familial status. The Baccigerinae is promoted under the senior synonym Faustulidae Poche, 1926, and the Echinobrevicecinae Dronen, Blend & McEachran, 1994 is synonymised with the Faustulidae. Consequently, species that were formerly in the Fellodistomidae are now distributed in three families: Fellodistomidae; Faustulidae (syn. Baccigerinae Yamaguti, 1954); and Tandanicolidae Johnston, 1927. We infer that the use of bivalves as intermediate hosts by this broad range of families indicates multiple host-switching events within the radiation of the Digenea.     

A review of the Apocreadiidae Skrjabin, 1942 (Trematoda: Digenea) and description of Australian species

The Apocreadiidae is reviewed and is considered to include genera recognised previously within the families Apocreadiidae, Homalometridae, Schistorchiidae, Sphincterostomatidae and Trematobrienidae. Key features of the family are extensive vitelline follicles, eye-spot pigment dispersed in forebody, I-shaped excretory vesicle, no cirrus-sac and genital pore opening immediately anterior to the ventral sucker (usually) or immediately posterior to it (Postporus Manter, 1949). Three subfamilies and 18 genera are recognised within the Apocreadiidae. The Apocreadiinae comprises Homalometron Stafford, 1904 (new syn. Barbulostomum Ramsey, 1965), Callohelmis n. g., Choanodera Manter, 1940, Crassicutis Manter, 1936, Dactylotrema Bravo-Hollis & Manter, 1957, Marsupioacetabulum Yamaguti, 1952, Microcreadium Simer, 1929, Myzotus Manter, 1940, Neoapocreadium Siddiqi & Cable, 1960, Neomegasolena Siddiqi & Cable, 1960, Pancreadium Manter, 1954, Procaudotestis Szidat, 1954 and Trematobrien Dollfus, 1950. The Schistorchiinae comprises Schistorchis Lühe, 1906, Sphincterostoma Yamaguti, 1937, Sphincteristomum Oshmarin, Mamaev & Parukhin, 1961 and Megacreadium Nagaty, 1956. The Postporinae comprises only Postporus. A key to subfamilies and genera of the Apocreadiidae is provided. It is argued that there is no convincing basis for the recognition of the genus Apocreadium Manter, 1937 and all its constituent species are combined with Homalometron. The following new combinations are proposed for species previously recognised within Apocreadium: Homalometron balistis (Manter, 1947), H. caballeroi (Bravo-Hollis, 1953), H. cryptum (Overstreet, 1969), H. longisinosum (Manter, 1937), H. manteri (Overstreet, 1970), H. mexicanum (Manter, 1937) and H. vinodae (Ahmad, 1985). Apocreadium uroproctoferum Sogandares-Bernal, 1959 is found to lack a uroproct and is made a synonym of H. mexicanum. Homalometron verrunculi nom. nov. is proposed to replace the secondarily pre-occupied H. caballeroi Lamothe-Argumedo, 1965. Barbulostomum is made a synonym of Homalometron and H. cupuloris (Ramsey, 1965) n. comb. is proposed. Neochoanodera is made a synonym of Choanodera and Choanodera ghanensis (Fischthal & Thomas, 1970) n. comb. is proposed. Species within the Apocreadiinae and Postporinae are reviewed and the following are recorded or described from Australian fishes: Homalometron wrightae n. sp. from Achlyopa nigra (Macleay), H. synagris (Yamaguti, 1953) n. comb. from Scolopsis monogramma (Cuvier), H. stradbrokensis n. sp. from Gerres subfasciatus Cuvier, Marsupioacetabulum opallioderma n. sp. from G. subfasciatus, Neoapocreadium karwarensis (Hafeezullah, 1970) n. comb. from G. subfasciatus, N. splendens n. sp. from S. monogramma and Callohelmis pichelinae n. g., n. sp. from Hemigymnus melapterus (Bloch), H. fasciatus (Bloch), Stethojulis bandanensis (Bleeker) andChoerodon venustus (De Vis). Callohelmis is recognised by the combination of absence of tegumental spines, caeca terminating midway between the testes and posterior end of body, ventral sucker enclosed in a tegumental pouch, prominent muscles radiating through the body from the ventral sucker, vitelline follicles not extending into the forebody, and a very short excretory vesicle that opens ventrally. New combinations for species previously recognised within Crassicutis are proposed as follows: Neoapocreadium caranxi (Bilqees, 1976) n. comb., N. gerridis (Nahhas & Cable, 1964) n. comb., N. imtiazi (Ahmad, 1984) n. comb. and N. marina (Manter, 1947) n. comb. The host-specificity and zoogeography of the Apocreadiinae are considered.     

Phylogeny and classification of the Digenea (Platyhelminthes: Trematoda)

Complete small subunit ribosomal RNA gene (ssrDNA) and partial (D1-D3) large subunit ribosomal RNA gene (lsrDNA) sequences were used to estimate the phylogeny of the Digenea via maximum parsimony and Bayesian inference. Here we contribute 80 new ssrDNA and 124 new lsrDNA sequences. Fully complementary data sets of the two genes were assembled from newly generated and previously published sequences and comprised 163 digenean taxa representing 77 nominal families and seven aspidogastrean outgroup taxa representing three families. Analyses were conducted on the genes independently as well as combined and separate analyses including only the higher plagiorchiidan taxa were performed using a reduced-taxon alignment including additional characters that could not be otherwise unambiguously aligned. The combined data analyses yielded the most strongly supported results and differences between the two methods of analysis were primarily in their degree of resolution. The Bayesian analysis including all taxa and characters, and incorporating a model of nucleotide substitution (general-time-reversible with among-site rate heterogeneity), was considered the best estimate of the phylogeny and was used to evaluate their classification and evolution. In broad terms, the Digenea forms a dichotomy that is split between a lineage leading to the Brachylaimoidea, Diplostomoidea and Schistosomatoidea (collectively the Diplostomida nomen novum (nom. nov.)) and the remainder of the Digenea (the Plagiorchiida), in which the Bivesiculata nom. nov. and Transversotremata nom. nov. form the two most basal lineages, followed by the Hemiurata. The remainder of the Plagiorchiida forms a large number of independent lineages leading to the crown clade Xiphidiata nom. nov. that comprises the Allocreadioidea, Gorgoderoidea, Microphalloidea and Plagiorchioidea, which are united by the presence of a penetrating stylet in their cercariae. Although a majority of families and to a lesser degree, superfamilies are supported as currently defined, the traditional divisions of the Echinostomida, Plagiorchiida and Strigeida were found to comprise non-natural assemblages. Therefore, the membership of established higher taxa are emended, new taxa erected and a revised, phylogenetically based classification proposed and discussed in light of ontogeny, morphology and taxonomic history.     

A new species of Dactylostomum Woolcock, 1935 (Digenea: Opecoelidae) from the goatfish Upeneichthys lineatus (Bloch & Schneider) (Mullidae) in Western Australian waters

Dactylostomum nicolli n. sp. is described from the intestine of the goatfish Upeneichthys lineatus (Bloch & Schneider) (Mullidae) caught off Point Peron, Western Australia. The problem of assigning the new species to the appropriate genus, especially with regard to its close resemblance to Paropecoelus Pritchard, 1966, which also infects goatfishes, is discussed. This is the second report of Dactylostomum Woolcock, 1935 from an Australian marine fish. The new species is distinguished by its elongate form, simple ventral sucker papillae and irregularly shaped ovary.