A family of textilinin genes, two of which encode proteins with antihaemorrhagic properties

Two peptides, textilinins 1 and 2, isolated from the venom of the Australian common brown snake, Pseudonaja textilis textilis, are effective in preventing blood loss. To further investigate the potential of textilinins as anti-haemorrhagic agents, we cloned cDNAs encoding these proteins. The isolated full-length cDNA (430 bp in size) was shown to code for a 59 amino acid protein, corresponding in size to the native peptide, plus an additional 24 amino acid propeptide. Six such cDNAs were identified, differing in nucleotide sequence in the coding region but with an identical propeptide. All six sequences predicted peptides containing six conserved cysteines common to Kunitz-type serine protease inhibitors. When expressed as glutathione S-transferase (GST) fusion proteins and released by cleavage with thrombin, only those peptides corresponding to textilinin 1 and 2 were active in inhibiting plasmin with K-i values similar to those of their native counterparts and in binding to plasmin less tightly than aprotinin by two orders of magnitude. Similarly, in the mouse tail vein blood loss model only recombinant textilinin 1 and 2 were effective in reducing blood loss. These recombinant textilinins have potential as therapeutic agents for reducing blood loss in humans, obviating the need for reliance on aprotinin, a bovine product with possible risk of transmissible disease, and compromising the fibrinolytic system in a less irreversible manner.

Is Cadinella ornatissima a chomodorid? Sperm ultra structure in an enigmatic nudibranch (Opisthobranchia Mollusca)

Sperm ultrastructure is described for the nudibranch gastropod Cadlinella ornatissima, type species of the genus Cadlinella (Thiele). Although C. ornatissima exhibits most of the sperm features characteristic of other Opisthobranchia and the Pulmonata (a small, rounded acrosomal vesicle, a complex, helical, mitochondrial derivative - partially paracrystalline, coarse fibres associated with the axoneme), it also possesses a number of previously undescribed and possibly unique features (a longitudinally inrolled acrosomal pedestal, an axial structure within the cavity of the acrosomal pedestal, an electron-dense collar at the anterior region of the acrosomal pedestal, the presence of crystalloid bodies within the glycogen helices of the mitochondrial derivative). To our knowledge this is the first report of crystalloid bodies in mature sperm of any mollusc. Collectively this evidence raises questions concerning the affinities and systematic position of Cadlinella within the Nudibranchia. The peculiar nature of the sperm differences, in comparison with other investigated nudibranchs, suggest that Cadlinella is not easily linked to either the Cadlinidae or Chromodorididae, and should be considered incertae sedis.

Mitochondrial genomes of parasitic flatworms

Complete or near-complete mitochondrial genomes are now available for 11 species or strains of parasitic flatworms belonging to the Trematoda and the Cestoda. The organization of these genomes is not strikingly different from those of other eumetazoans, although one gene (atp8) commonly found in other phyla is absent from flatworms. The gene order in most flatworms has similarities to those seen in higher protostomes such as annelids. However, the gene order has been drastically altered in Schistosoma mansoni, which obscures this possible relationship. Among the sequenced taxa, base composition varies considerably, creating potential difficulties for phylogeny reconstruction. Long non-coding regions are present in all taxa, but these vary in length from only a few hundred to similar to10 000 nucleotides. Among Schistosoma spp., the long non-coding regions are rich in repeats and length variation among individuals is known. Data from mitochondrial genomes are valuable for studies on species identification, phylogenies and biogeography.

Prolactin-releasing peptide in ewe: cDNA cloning, mRNA distribution and effects on prolactin secretion in vitro and in vivo

RT-PCR followed by 5'- and 3'- rapid amplification of cDNA ends was used to clone and sequence ovine prolactin-releasing peptide (PrRP). The cDNA was characterised by short 5'- and 3'-untranslated regions and a GC-rich (71%) coding region. The nucleotide and deduced amino acid sequences for the coding region showed 95.6 and 94.9% identity with bovine PrRP but the amino acid sequence of PrRP31 was conserved between these species. Northern blot analysis and RT-PCR showed that, as in the rat, the peptide was more abundantly expressed in the brainstem than the hypothalamus. However, in the ovine hypothalamus, PrRP mRNA expression was more widespread than in the rat, with expression detected in both rostral and caudal parts of the mediobasal hypothalamus. The effects of synthetic ovine PrRP on prolactin secretion both in vitro and in vivo were also examined. In primary cultures of sheep pituitary cells, PrRP significantly (P

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

Complete mitochondrial genomes confirm the distinctiveness of the horse-dog and sheep-dog strains of Echinococcus granulosus

Unlike other members of the genus, Echinococcus granulosus is known to exhibit considerable levels of variation in biology, physiology and molecular genetics. Indeed, some of the taxa regarded as 'genotypes' within E. granulosus might be sufficiently distinct as to merit specific status. Here, complete mitochondrial genomes are presented of 2 genotypes of E. granulosus (G1-sheep-dog strain: G4-horse-dog strain) and of another taeniid cestode, Taenia crassiceps. These genomes are characterized and compared with those of Echinococcus multilocularis and Hymenolepis diminuta. Genomes of all the species are very similar in structure, length and base-composition. Pairwise comparisons of concatenated protein-coding genes indicate that the G1 and G4 genotypes of E. granulosus are almost as distant from each other as each is from a distinct species, E. multilocularis. Sequences for the variable genes atp6 and nad3 were obtained from additional genotypes of E. granulosus, from E. vogeli and E. oligarthrus. Again, pairwise comparisons showed the distinctiveness of the G1 and G4 genotypes. Phylogenetic analyses of concatenated atp6, nad1 (partial) and cox1 (partial) genes from E. multilocularis, E. vogeli, E. oligarthrus, 5 genotypes of E. granulosus, and using T. crassiceps as an outgroup, yielded the same results. We conclude that the sheep-dog and horse-dog strains of E. granulosus should be regarded as distinct at the specific level.

Reverse transcriptase activity and untranslated region sharing of a new RTE-like, non-long terminal repeat retrotransposon from the human blood fluke, Schistosoma japonicum

A new RTE-like, non-long terminal repeat retrotransposon, termed SjR2, from the human blood fluke, Schistosoma japonicum, is described. SjR2 is similar to3.9 kb in length and is constituted of a single open reading frame encoding a polyprotein with apurinic/apyrimidinic endonuclease and reverse transcriptase domains. The open reading frame is bounded by 5'- and 3'-terininal untranslated regions and, at its 3-terminus, SjR2 bears a short (TGAC)(3) repeat. Phylogenetic analyses based on conserved domains of reverse transcriptase or endonuclease revealed that SjR2 belonged to the RTE clade of non-long terminal repeat retrotransposons. Further, SjR2 was homologous, but probably not orthologous, to SR2 front the African blood fluke, Schistosoma mansoni; this RTE-like family of non-long terminal repeat retrotransposons appears to have arisen before the divergence of the extant schistosome species. Hybridisation analyses indicated that similar to 10,000 copies of SjR2 were dispersed throughout the S. japonicum chromosomes, accounting for up to 14% of the nuclear genome. Messenger RNAs encoding the reverse transcriptase and endonuclease domains of SjR2 were detected in several developmental stages of the schistosome, indicating that the retrotransposon was actively replicating within the genome of the parasite. Exploration of the coding and non-coding regions of SjR2 revealed two notable characteristics. First, the recombinant reverse transcriptase domain of SjR2 expressed in insect cells primed reverse transcription of SjR2 mRNA in vitro. By contrast, recombinant SjR2-endonuclease did not appear to cleave schistosome or plasmid DNA. Second, the 5'-untranslated region of SjR2 was >80% identical to the 3-untranslated region of a schistosome heat shock protein-70 gene (hsp-70) in the antisense orientation, indicating that SjR2-like elements were probably inserted into the non-coding regions of ancestral S. japonicum HSP-70, probably after the species diverged from S. mansoni. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Effects of polyunsaturated fatty acids on voltage-gated K+ and Na+ channels in rat olfactory receptor neurons

Although the polyunsaturated fatty acids arachidonic acid (AA) and docosahexaenoic acid (DHA) are enriched in the olfactory mucosa, their possible contribution to olfactory transduction has not been investigated. This study characterized their effects on voltage-gated K+ and Na+ channels of rat olfactory receptor neurons. Physiological (3-10 mum) concentrations of AA and DHA potently and irreversibly inhibited the voltage-gated K+ current in a voltage-independent manner. In addition, both compounds significantly reduced the inhibitory potency of the odorants acetophenone and amyl acetate at these channels. By comparison, the steady-state effects of both AA and DHA on the voltage-gated Na+ channel were relatively weak, with half-maximal inhibition requiring approximate to 35 mum of either compound. However, a surprising finding was that the initial application of 3 mum AA to a naive neuron caused a strong but transient inhibition of the Na+ current. The channels became almost completely resistant to this inhibition within 1 min, and a 2-min wash in control solution was insufficient to restore the strong inhibitory effect. These observations suggest that polyunsaturated fatty acids have the potential to strongly influence the coding of odorant information by olfactory receptor neurons.

Isolation and characterisation of a novel rabbit sulfotransferase isoform belonging to the SULT1A subfamily

Sulfotransferases (SULTs) catalyse the sulfonation of both endogenous and exogenous compounds including hormones, catecholamines. drugs and xenobiotics. While in most occasions, sulfonation is a detoxication pathway. in the case of certain drugs and carcinogens. it leads to metabolic activation. Since, the rabbit has been extensively used for both pharmacological and toxicological studies, the purpose of this study was to further characterise the sulfotransferase system of this animal. In the present study, a novel sulfotransferase isoform (GenBank Accession no. AF360872) was isolated from a rabbit liver cDNA lambdaZAP 11 library. The full-length sequence of the clone was 1138 bp long and contained a coding region of 888 bp encoding a cytosolic protein of 295 amino acids (deduced molecular weight 34,193 Da). The amino acid sequence of this novel SULT isoform showed >70% identity with members of the SULT1A subfamily of sulfotransferases from other species. Upon expression of the encoded rabbit sulfotransferase in Escherchia coli (E. coli), it was shown that the enzyme was capable of sulfonating both p-nitrophenot (K-m and V-max values of 0.15 muM and 897.5 nmol/min/mg protein. respectively) and dopamine (K-m and V-max values of 175.3 muM and 151.1 nmol/min/mg protein, respectively). Based on the sequence data obtained and substrate specificity, this new rabbit sulfotransferase was named rabSULT1A1. Immunoblotting was used to demonstrate that rabSULT1A1 protein is expressed in liver, duodenum, jejunum, ileum, colon and recturm. (C) 2002 Elsevier Science Ltd. All rights reserved.

Association of the SULT1A1 R213H polymorphism with colorectal cancer

1. Sulphotransferases are a superfamily of enzymes involved in both detoxification and bioactivation of endogenous and exogenous compounds. The arylsulphotransferase SULT1A1 has been implicated in a decreased activity and thermostability when the wild-type arginine at position 213 of the coding sequence is substituted by a histidine. SULT1A1 is the isoform primarily associated with the conversion of dietary N -OH arylamines to DNA binding adducts and is therefore of interest to determine whether this polymorphism is linked to colorectal cancer. 2. Genotyping, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, was performed using DNA samples of healthy control subjects (n = 402) and patients with histologically proven colorectal cancer (n = 383). Both control and test populations possessed similar frequencies for the mutant allele (32.1 and 31%, respectively; P = 0.935). Results were not altered when age and gender were considered as potential confounders in a logistic regression analysis. 3. Examination of the sulphonating ability of the two allozymes with respect to the substrates p -nitrophenol and paracetamol showed that the affinity and rate of sulphonation was unaffected by substitution of arginine to histidine at position 213 of the amino acid sequence. 4. From this study, we conclude that the SULT1A1 R213H polymorphism is not linked with colorectal cancer in this elderly Australian population.

An investigation into the possible inhibitory effects of white cypress pine (Callitris glaucophylla) litter on the germination and growth of associated ground cover species

White cypress-pine stands typically support sparse densities of shrubs and grasses. The commonly held opinion is that leaching of allelopathic chemical compounds from cypress-pine litter partly facilitates this exclusion. Germination and growth of cypress pine seedlings do not appear to be similarly affected. This study set out to determine whether cypress litter had a differential effect on germination and growth of cypress-pine seedlings and on associated ground-cover species. Glasshouse trials comparing seedling emergence under cypress- and artificial-litter layers were undertaken. Cypress-pine litter did not have an inhibitory effect on the germination or growth of ground-cover species. In most cases, seedling emergence was facilitated by the application of cypress-pine litter due to its ability to increase the water holding capacity of the underlying soil. Cypress litter did not promote growth of its own seedlings over its competitors except on coarse-textured soils where it provided an ameliorative function to water stress due to the soil's reduced water holding capacity. The inhibition of ground-cover species' germination and growth in pure cypress stands was suggested to be the result of high below-ground resource competition due to the pine's expansive root morphology.

Quantitation of alternatively spliced NMDA receptor NR1 isoform mRNA transcripts in human brain by competitive RT-PCR

The N-methyl-D-aspartate (NMDA)-selective subtype of ionotropic glutamate receptor is of importance in neuronal differentiation and synapse consolidation, activity-dependent forms of synaptic plasticity, and excitatory amino acid-mediated neuronal toxicity [Neurosci. Res. Program, Bull. 19 (1981) 1; Lab. Invest. 68 (1993) 372]. NMDA receptors exist in vivo as tetrameric or pentameric complexes comprising proteins from two families of homologous subunits, designated NR1 and NR2(A-D) [Biochem. Biophys. Res. Commun. 185 (1992) 826]. The gene coding for the human NR1 subunit (hNR1) is composed of 21 exons, three of which (4, 20 and 21) can be differentially spliced to generate a total of eight distinct subunit variants. We detail here a competitive RT-PCR (cRT-PCR) protocol to quantify endogenous levels of hNR1 splice variants in autopsied human brain. Quantitation of each hNR1 splice variant is performed using standard curve methodology in which a known amount of synthetic ribonucleic acid competitor (internal standard) is co-amplified against total RNA. This method can be used for the quantitation of hNR1 mRNA levels in response to acute or chronic disease states, in particular in the glutamatergic-associated neuronal loss observed in Alzheimer's disease [J. Neurochem. 78 (2001) 175]. Furthermore, alterations in hNR1 mRNA expression may be reflected at the translational level, resulting in functional changes in the NMDA receptor. (C) 2003 Elsevier Science B.V. All rights reserved.

Critical comparison of Julius Kruttschnitt Mineral Research Centre (JKMRC) blast fragmentation models

Blast fragmentation can have a significant impact on the profitability of a mine. An optimum run of mine (ROM) size distribution is required to maximise the performance of downstream processes. If this fragmentation size distribution can be modelled and controlled, the operation will have made a significant advancement towards improving its performance. Blast fragmentation modelling is an important step in Mine to Mill™ optimisation. It allows the estimation of blast fragmentation distributions for a number of different rock mass, blast geometry, and explosive parameters. These distributions can then be modelled in downstream mining and milling processes to determine the optimum blast design. When a blast hole is detonated rock breakage occurs in two different stress regions - compressive and tensile. In the-first region, compressive stress waves form a 'crushed zone' directly adjacent to the blast hole. The second region, termed the 'cracked zone', occurs outside the crush one. The widely used Kuz-Ram model does not recognise these two blast regions. In the Kuz-Ram model the mean fragment size from the blast is approximated and is then used to estimate the remaining size distribution. Experience has shown that this model predicts the coarse end reasonably accurately, but it can significantly underestimate the amount of fines generated. As part of the Australian Mineral Industries Research Association (AMIRA) P483A Mine to Mill™ project, the Two-Component Model (TCM) and Crush Zone Model (CZM), developed by the Julius Kruttschnitt Mineral Research Centre (JKMRC), were compared and evaluated to measured ROM fragmentation distributions. An important criteria for this comparison was the variation of model results from measured ROM in the-fine to intermediate section (1-100 mm) of the fragmentation curve. This region of the distribution is important for Mine to Mill™ optimisation. The comparison of modelled and Split ROM fragmentation distributions has been conducted in harder ores (UCS greater than 80 MPa). Further work involves modelling softer ores. The comparisons will be continued with future site surveys to increase confidence in the comparison of the CZM and TCM to Split results. Stochastic fragmentation modelling will then be conducted to take into account variation of input parameters. A window of possible fragmentation distributions can be compared to those obtained by Split . Following this work, an improved fragmentation model will be developed in response to these findings.

A partial velocity approach to subcycling structural dynamics

Subcycling, or the use of different timesteps at different nodes, can be an effective way of improving the computational efficiency of explicit transient dynamic structural solutions. The method that has been most widely adopted uses a nodal partition. extending the central difference method, in which small timestep updates are performed interpolating on the displacement at neighbouring large timestep nodes. This approach leads to narrow bands of unstable timesteps or statistical stability. It also can be in error due to lack of momentum conservation on the timestep interface. The author has previously proposed energy conserving algorithms that avoid the first problem of statistical stability. However, these sacrifice accuracy to achieve stability. An approach to conserve momentum on an element interface by adding partial velocities is considered here. Applied to extend the central difference method. this approach is simple. and has accuracy advantages. The method can be programmed by summing impulses of internal forces, evaluated using local element timesteps, in order to predict a velocity change at a node. However, it is still only statistically stable, so an adaptive timestep size is needed to monitor accuracy and to be adjusted if necessary. By replacing the central difference method with the explicit generalized alpha method. it is possible to gain stability by dissipating the high frequency response that leads to stability problems. However. coding the algorithm is less elegant, as the response depends on previous partial accelerations. Extension to implicit integration, is shown to be impractical due to the neglect of remote effects of internal forces acting across a timestep interface. (C) 2002 Elsevier Science B.V. All rights reserved.

Stratigraphy, facies architecture and tectonic implications of the Upper Devonian to Lower Carboniferous Campwyn Volcanics of the northern New England Fold Belt

Upper Devonian to Lower Carboniferous strata of the Campwyn Volcanics of east central Queensland preserve a substantial sequence of first-cycle volcaniclastic sedimentary and coeval volcanic rocks that record prolonged volcanic activity along the northern New England Fold Belt. The style and scale of volcanism varied with time, producing an Upper Devonian sequence of mafic volcano-sedimentary rocks overlain by a rhyolitic ignimbrite-dominated sequence that passes upward into a Lower Carboniferous limestone-bearing sedimentary sequence. We define two facies associations for the Campwyn Volcanics. A lower facies association is dominated by mafic volcanic-derived sedimentary breccias with subordinate primary mafic volcanic rocks comprising predominantly hyaloclastite and peperite. Sedimentary breccias record episodic and high energy, subaqueous depositional events with clastic material sourced from a mafic lava-dominated terrain. Some breccias contain a high proportion of attenuated dense, glassy mafic juvenile clasts, suggesting a syn-eruptive origin. The lower facies association coarsens upwards from a lithic sand-dominated sequence through a thick interval of pebble- to boulder-grade polymict volcaniclastic breccias, culminating in facies that demonstrate subaerial exposure. The silicic upper facies association marks a significant change in eruptive style, magma composition and the nature of eruptive sources, as well as the widespread development of subaerial depositional conditions. Crystal-rich, high-grade, low- to high-silica rhyolite ignimbrites dominate the base of this facies association. Biostratigraphic age controls indicate that the ignimbrite-bearing sequences are Famennian to lower-mid Tournaisian in age. The ignimbrites represent extra-caldera facies with individual units up to 40 m thick and mostly lacking coarse lithic breccias. Thick deposits of pyroclastic material interbedded with fine-grained siliceous sandstone and mudstone (locally radiolarian-bearing) were deposited from pyroclastic flows that crossed palaeoshorelines or represent syn-eruptive, resedimented pyroclastic material. Some block-bearing lithic-pumice-crystal breccias may also reflect more proximal subaqueous silicic explosive eruptions. Crystal-lithic sandstones interbedded with, and overlying the ignimbrites, contain abundant detrital volcanic quartz and feldspar derived from the pyroclastic deposits. Limestone is common in the upper part of the upper facies association, and several beds are oolitic (cf. Rockhampton Group of the Yarrol terrane). Overall, the upper facies association fines upward and is transgressive, recording a return to shallow-marine conditions. Palaeocurrent data from all stratigraphic levels in the Campwyn Volcanics indicate that the regional sediment-dispersal direction was to the northwest, and opposed to the generally accepted notion of easterly sediment dispersal from a volcanic arc source. The silicic upper facies association correlates in age and lithology to Early Carboniferous silicic volcanism in the Drummond (Cycle 1) and Burdekin Basins, Connors Arch, and in the Yarrol terranes of eastern Queensland. The widespread development of silicic volcanism in the Early Carboniferous indicates that silicic (rift-related) magmatism was not restricted to the Drummond Basin, but was part of a more substantial silicic igneous province.