Spatial distribution of benthic microalgae on coral reefs determined by remote sensing

Understanding the ecological role of benthic microalgae, a highly productive component of coral reef ecosystems, requires information on their spatial distribution. The spatial extent of benthic microalgae on Heron Reef (southern Great Barrier Reef, Australia) was mapped using data from the Landsat 5 Thematic Mapper sensor. integrated with field measurements of sediment chlorophyll concentration and reflectance. Field-measured sediment chlorophyll concentrations. 2 ranging from 23-1.153 mg chl a m(2), were classified into low, medium, and high concentration classes (1-170, 171-290, and > 291 mg chl a m(-2)) using a K-means clustering algorithm. The mapping process assumed that areas in the Thematic Mapper image exhibiting similar reflectance levels in red and blue bands would correspond to areas of similar chlorophyll a levels. Regions of homogenous reflectance values corresponding to low, medium, and high chlorophyll levels were identified over the reef sediment zone by applying a standard image classification algorithm to the Thematic Mapper image. The resulting distribution map revealed large-scale ( > 1 km 2) patterns in chlorophyll a levels throughout the sediment zone of Heron Reef. Reef-wide estimates of chlorophyll a distribution indicate that benthic Microalgae may constitute up to 20% of the total benthic chlorophyll a at Heron Reef. and thus contribute significantly to total primary productivity on the reef.

Inducible system in human hepatoma cell lines for hepatitis C virus production

We cloned the complete complementary DNA of an isolate of the hepatitis C virus, HCV-S1, into a tetra cycline-inducible expression vector and stably transfected it into two human hepatoma cell lines, Huh7 and HepG2. Twenty-six Huh7 and two HepG2-positive clones were obtained after preliminary screening. Two Huh7 (SH-7 and -9) and one HepG2 (G-19) clones were chosen for further characterisation. Expression of HCV proteins in these cells accumulated from 6 In to 4 days posttreatment. Full-length viral plus-strand RNA was detected by Northern analyses. Using RT-PCR and ribonuclease protection assay, we also detected the synthesis of minus-strand HCV RNA. Plus- and minus-strand viral RNA was still detected after treatment with actinomycin D. Indirect immunofluorescence staining with anti-E2, NS4B, and NS5A revealed that these proteins were mostly localised to the endoplasmic reticulum (ER). Culture media from tet-induced SH-9 cells was separated on sucrose density gradients and analysed for the presence of HCV RNA. Viral RNA levels peaked at two separate ranges, one with a buoyant density of 1.08 g/ml and another from 1.17 to 1.39 g/ml. Electron microscopy demonstrated the presence of subviral-like particles (approximately 20-25 nm in diameter) in the cytoplasm of SH-9 and G-19 cells, which were positively labelled by anti-HCV core antibodies. Anti-E2 antibodies strongly labelled cytoplasmic vesicular structures and some viral-like particles. Complete viral particles of about 50 nm which reacted with anti-E2 antibodies were observed in the culture media of tet-induced SH-9 cells following negative staining. Supernatant from tet-treated SH-9 cells was found to infect naive Huh7 and stable Huh7-human CD81 cells. (C) 2002 Elsevier Science (USA).

Explicit evidence-based prescribing criteria - an important step in achieving quality therapeutics in nursing homes

Reaction between 5-(4-amino-2-thiabutyl)-5-methyl-3,7-dithianonane-1, 9-diamine (N3S3) and 5- methyl-2,2-bipyridine-5-carbaldehyde and subsequent reduction of the resulting imine with sodium borohydride results in a potentially ditopic ligand (L). Treatment of L with one equivalent of an iron( II) salt led to the monoprotonated complex [Fe(HL)](3+), isolated as the hexafluorophosphate salt. The presence of characteristic bands for the tris( bipyridyl) iron( II) chromophore in the UV/vis spectrum indicated that the iron( II) atom is coordinated octahedrally by the three bipyridyl (bipy) groups. The [Fe( bipy) 3] moiety encloses a cavity composed of the N3S3 portion of the ditopic ligand. The mononuclear and monomeric nature of the complex [Fe(HL)](3+) has been established also by accurate mass analysis. [Fe(HL)](3+) displays reduced stability to base compared with the complex [Fe(bipy)(3)](2+). In aqueous solution [Fe(HL)](3+) exhibits irreversible electrochemical behaviour with an oxidation wave ca. 60 mV to more positive potential than [Fe(bipy)(3)](2+). Investigations of the interaction of [Fe(L)](2+) with copper( II), iron( II), and mercury( II) using mass spectroscopic and potentiometric methods suggested that where complexation occurred, fewer than six of the N3S3 cavity donors were involved. The high affinity of the complex [Fe(L)](2+) for protons is one reason suggested to contribute to the reluctance to coordinate a second metal ion.

Heterogeneous nuclear ribonucleoprotein A3, a novel RNA trafficking response element-binding protein

The cis-acting response element, A2RE, which is sufficient for cytoplasmic mRNA trafficking in oligodendrocytes, binds a small group of rat brain proteins. Predominant among these is heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor for cytoplasmic trafficking of RNAs bearing A2RE-like sequences. We have now identified the other A2RE-binding proteins as hnRNP A1/A1(B), hnRNP B1, and four isoforms of hnRNP A3. The rat and human hnRNP A3 cDNAs have been sequenced, revealing the existence of alternatively spliced mRNAs. In Western blotting, 38-, 39-, 41 -, and 41.5-kDa components were all recognized by antibodies against a peptide in the glycine-rich region of hnRNP A3, but only the 41- and 41.5-kDa bands bound antibodies to a 15-residue N-terminal peptide encoded by an alternatively spliced part of exon 1. The identities of these four proteins were verified by Edman sequencing and mass spectral analysis of tryptic fragments generated from electrophoretically separated bands. Sequence-specific binding of bacterially expressed hnRNP A3 to A2RE has been demonstrated by biosensor and UV cross-linking electrophoretic mobility shift assays. Mutational analysis and confocal microscopy data support the hypothesis that the hnRNP A3 isoforms have a role in cytoplasmic trafficking of RNA.

Cenozoic continental weathering and its implications for the palaeoclimate: evidence from Ar-40/Ar-39 geochronology of supergene K-Mn oxides in Mt Tabor, central Queensland, Australia

One hundred and twenty-five mineral grains from 45 visually pure K-bearing Mn oxide (hollandite group) samples collected from weathering profiles in the Mt Tabor region of central Queensland, Australia, were analysed by the Ar-40/Ar-39 laser probe technique. These K-Mn oxides precipitated mainly through a process of cavity filling (direct precipitation from weathering solution), with botryoidal texture formed by micrometric mineral bands. Well-defined and reproducible plateau ages have been obtained for most samples, ranging from 27.2 +/- 0.8 to 6.8 +/- 0.5 Ma (2 sigma). Statistical analysis of the geochronological results by mixture modelling suggests an episodic mineral precipitation history, with two major peaks at 20.2 +/- 0.22 Ma and 16.5 +/- 0.17 Ma. The geochronological results, when combined with information on paragenetic relationships and mineralogical textures obtained from petrographic, scanning electron microscopy, and electron microprobe investigations, indicate that warm and humid palaeoclimatic conditions favourable to intense chemical weathering prevailed in central Queensland from late Oligocene to middle Miocene, particularly in the early Miocene. These results, in conjunction with previous and ongoing investigations in NW and eastern Queensland, suggest that most of Queensland was dominated by humid climates during the Miocene. (C) 2002 Elsevier Science BN. All rights reserved.

Evolution of the secondary structure of the rRNA internal transcribed spacer 2 (ITS2) in hard ticks (Ixodidae, Arthropoda)

ITS2 sequences are used extensively in molecular taxonomy and population genetics of arthropods and other animals yet little is known about the molecular evolution of ITS2. We studied the secondary structure of ITS2 in species from each of the six main lineages of hard ticks (family Ixodidae). The ITS2 of these ticks varied in length from 679 bp in Ixodes scapularis to 1547 bp in Aponomma concolor. Nucleotide content varied also: the ITS2 of ticks from the Prostriata lineage (Ixodes spp.) had 46-49% GC whereas ITS2 sequences of ticks from the Metastriata lineage (all other hard ticks) had 61-62% GC. Despite variation in nucleotide sequence, the secondary structure of the ITS2 of all of these ticks apparently has five domains. Stems 1, 3, 4 and 5 of this secondary structure were obvious in all of the species studied. However, stem 2 was not always obvious despite the fact that it is flanked by highly conserved sequence motifs in the adjacent stems, stems 1 and 3. The ITS2 of hard ticks has apparently evolved mostly by increases and decreases in length of the nucleotide sequences, which caused increases, and decreases in the length of stems of the secondary structure. This is most obvious when stems of the secondary structures of the Prostriata (Ixodes spp.) are compared to those of the Metastriata (all other hard ticks). Increases in the size of the ITS2 may have been caused by replication slippage which generated large repeats, like those seen in Haemaphysalis humerosa and species from the Rhipicepalinae lineage, and the small repeats found in species from the other lineages of ticks.

Analysis of genetic diversity within Australian lucerne cultivars and implications for future genetic improvement

Lucerne (Medicago sativa L.) is autotetraploid, and predominantly allogamous. This complex breeding structure maximises the genetic diversity within lucerne populations making it difficult to genetically discriminate between populations. The objective of this study was to evaluate the level of random genetic diversity within and between a selection of Australian-grown lucerne cultivars, with tetraploid M. falcata included as a possible divergent control source. This diversity was evaluated using random amplified polymorphic DNA (RAPDs). Nineteen plants from each of 10 cultivars were analysed. Using 11 RAPD primers, 96 polymorphic bands were scored as present or absent across the 190 individuals. Genetic similarity estimates (GSEs) of all pair-wise comparisons were calculated from these data. Mean GSEs within cultivars ranged from 0.43 to 0.51. Cultivar Venus (0.43) had the highest level of intra-population genetic diversity and cultivar Sequel HR (0.51) had the lowest level of intra-population genetic diversity. Mean GSEs between cultivars ranged from 0.31 to 0.49, which overlapped with values obtained for within-cultivar GSE, thus not allowing separation of the cultivars. The high level of intra- and inter-population diversity that was detected is most likely due to the breeding of synthetic cultivars using parents derived from a number of diverse sources. Cultivar-specific polymorphisms were only identified in the M. falcata source, which like M. sativa, is outcrossing and autotetraploid. From a cluster analysis and a principal components analysis, it was clear that M. falcata was distinct from the other cultivars. The results indicate that the M. falcata accession tested has not been widely used in Australian lucerne breeding programs, and offers a means of introducing new genetic diversity into the lucerne gene pool. This provides a means of maximising heterozygosity, which is essential to maximising productivity in lucerne.

Phenotypic and genotypic analysis of Helicoverpa armigera nucleopolyhedrovirus serially passaged in cell culture

Rapid accumulation of few polyhedra (FP) mutants was detected during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in cell culture. 100% FP infected cells were observed by passage 6. The specific yield decreased from 178 polyhedra per cell at passage 2 to two polyhedra per cell at passage 6. The polyhedra at passage 6 were not biologically active, with a 28-fold reduction in potency compared to passage 3. Electron microscopy studies revealed that very few polyhedra were produced in an FP infected cell (< 10 polyhedra per section) and in most cases these polyhedra contained no virions. A specific failure in the intranuclear nucleocapsid envelopment process in the FP infected cells, leading to the accumulation of naked nucleocapsids, was observed. Genomic restriction endonuclease digestion profiles of budded virus DNA from all passages did not indicate any large DNA insertions or deletions that are often associated with such FP phenotypes for the extensively studied Autographa californica nucleopolyhedrovirus and Gaileria mellonella nucleopolyhedrovirus. Within an HaSNPV 25K FP gene homologue, a single base-pair insertion (an adenine residue) within a region of repetitive sequences (seven adenine residues) was identified in one plaque-purified HaSNPV FP mutant. Furthermore, the sequences obtained from individual clones of the 25KFP gene PCR products of a late passage revealed point mutations or single base-pair insertions occurring throughout the gene. The mechanism of FP mutation in HaSNPV is likely similar to that seen for Lymantria dispar nucleopolyhedrovirus, involving point mutations or small insertions/deletions of the 25K FP gene.

Hepatic covalent adduct formation with zomepirac in the CD26-deficient mouse

Background and Aims: Zomepirac (ZP), a non-steroidal anti-inflammatory drug (NSAID), has been reported to cause immune-mediated liver injury. In vivo, ZP is metabolized to a chemically reactive acyl glucuronide conjugate (ZAG) which can undergo covalent adduct formation with proteins. Such acyl glucuronide-derived drug-protein adducts may be important in the development of immune and toxic responses caused by NSAID. We have shown using immunoabsorptions that the 110 kDa CD26 (dipeptidyl peptidase IV) is one of the hepatic target proteins for covalent modification by ZAG. In the present study, a CD26-deficient mouse strain was used to examine protein targets for covalent modification by ZP/metabolites in the liver. Methods and Results: The CD26-deficient phenotype was confirmed by immunohistochemistry, flow cytometry analysis, RT-PCR, enzyme assay and immunoblotting. Moreover, by using monoclonal antibody immunoblots, CD26 was not detected in the livers of ZP-treated CD26-deficient mice. Immunoblots using a polyclonal antiserum to ZP on liver from ZP-treated mice showed three major sizes of protein bands, in the 70, 110 and 140 kDa regions. Most, but not all, of the anti-ZP immunoreactivity in the 110 kDa region was absent from ZP-treated CD26-deficient mice. Conclusion: These data definitively showed that CD26 was a component of ZP-modified proteins in vivo. In addition, the data suggested that at least one other protein of approximately 110 kDa was modified by covalent adduct formation with ZAG. (C) 2002 Blackwell Science Asia Pty Ltd.

Mineral chemistry, whole-rock compositions, and petrogenesis of leg 176 gabbros: Data and discussion

We report mineral chemistry, whole-rock major element compositions, and trace element analyses on Hole 735B samples drilled and selected during Leg 176. We discuss these data, together with Leg 176 shipboard data and Leg 118 sample data from the literature, in terms of primary igneous petrogenesis. Despite mineral compositional variation in a given sample, major constituent minerals in Hole 735B gabbroic rocks display good chemical equilibrium as shown by significant correlations among Mg# (= Mg/[Mg+Fe2+]) of olivine, clinopyroxene, and orthopyroxene and An (=Ca/[Ca+Na]) of plagioclase. This indicates that the mineral assemblages olivine + plagioclase in troctolite, plagioclase + clinopyroxene in gabbro, plagioclases + clinopyroxene + olivine in olivine gabbro, and plagioclase + clinopyroxene + olivine + orthopyroxene in gabbronorite, and so on, have all coprecipitated from their respective parental melts. Fe-Ti oxides (ilmenite and titanomagnetite), which are ubiquitous in most of these rocks, are not in chemical equilibrium with olivine, clinopyroxene, and plagioclase, but precipitated later at lower temperatures. Disseminated oxides in some samples may have precipitated from trapped Fe-Ti–rich melts. Oxides that concentrate along shear bands/zones may mark zones of melt coalescence/transport expelled from the cumulate sequence as a result of compaction or filter pressing. Bulk Hole 735B is of cumulate composition. The most primitive olivine, with Fo = 0.842, in Hole 735B suggests that the most primitive melt parental to Hole 735B lithologies must have Mg# ≤ 0.637, which is significantly less than Mg# = 0.714 of bulk Hole 735B.

beta-strand mimicking macrocyclic amino acids: Templates for protease inhibitors with antiviral activity

New amino acids are reported in which component macrocycles are constrained to mimic tripeptides locked in a beta-strand conformation. The novel amino acids involve macrocycles functionalized with both an N- and a C-terminus enabling addition of appendages at either end to modify receptor affinity, selectivity, or membrane permeability. We show that the cycles herein are effective templates within inhibitors of HIV-1 protease. Eleven compounds originating from such bifunctionalized cyclic templates are potent inhibitors of HIV-1 protease (Ki 0.3-50 nM; pH 6.5, I = 0.1 M). Unlike normal peptides comprising amino acids, five of these macrocycle-containing compounds are potent antiviral agents with sub-micromolar potencies (IC50 170-900 nM) against HIV-1 replication in human MT2 cells. The most active antiviral agents are the most lipophilic, with calculated values of LogD(6.5) greater than or equal to 4. All molecules have a conformationally constrained 17-membered macrocyclic ring that has been shown to structurally mimic a tripeptide segment (Xaa)-(Val/Ile)-(Phe/Tyr) of a peptide substrate in the extended conformation. The presence of two trans amide bonds and a para-substituted aromatic ring prevents intramolecular hydrogen bonds and fixes the macrocycle in the extended conformation. Similarly constrained macrocycles may be useful templates for the creation of inhibitors for the many other proteins and proteases that recognize peptide beta-strands.

Cross-reactivity of Sydney funnel-web spider antivenom: neutralization of the in vitro toxicity of other Australian funnel-web (Atrax and Hadronyche) spider venoms

Australian funnel-web spiders are recognized as one of the most venomous spiders to humans world-wide. Funnel-web spider antivenom (FWS AV) reverses clinical effects of envenomation from the bite of Atrax robustus and a small number of related Hadronyche species. This study assessed the in vitro efficacy of FWS AV in neutralization of the effects of funnel-web spider venoms, collected from various locations along the eastern seaboard of Australia, in an isolated chick biventer cervicis nerve-muscle preparation. Venoms were separated by SDS-PAGE electrophoresis to compare protein composition and transblotted for Western blotting and incubation with FWS AV. SDS-PAGE of venoms revealed similar low and high molecular weight protein bands. Western blotting with FWS AV showed similar antivenom binding with protein bands in all the venoms tested. Male funnel-web spider venoms (7/7) and female venoms (5110) produced muscle contracture and fasciculation when applied to the nerve-muscle preparation. Venom effects were reversed by subsequent application of FWS AV or prevented by pretreatment of the preparation with antivenom. FWS AV appears to reverse the in vitro toxicity of a number of funnel-web spider venoms from the eastern seaboard of Australia. FWS AV should be effective in the treatment of envenomation from most, if not all, species of Australian funnel-web spiders. (C) 2001 Elsevier Science Ltd. All rights reserved.

Secondary structure of the third extracellular loop responsible for ligand selectivity of a mammalian gonadotropin-releasing hormone receptor

The extracellular loop 3 (ECL3) of the mammalian gonadotropin-releasing hormone receptor (GnRH-R) contains an acidic amino acid (Glu(301) in the mouse GnRH-R,) that confers agonist selectivity for Are in mammalian GnRH. It is proposed that a specific conformation of ECL3 is necessary to orientate the carboxyl side chain of the acidic residue for interaction with Arg(8) of GnRH, which is supported by decreased affinity for Arg(8) GnRH but not Gln(8) GnRH when an adjacent Pro is mutated to Ala. To probe the structural contribution of the loop domain to the proposed presentation of the carboxyl side chain, we synthesized a model peptide (CGPEMLNRVSEPGC) representing residues 293-302 of mouse ECL3, where Cys and Gly residues are added symmetrically at the N and C termini, respectively, allowing the introduction of a disulfide bridge to simulate the distances at which the ECL3 is tethered to the transmembrane domains 6 and 7 of the receptor. The ability of the ECL3 peptide to bind GnRH with low affinity was demonstrated by its inhibition of GnRH stimulation of inositol phosphate production in cells expressing the GnRH-R. The CD bands of the ECL3 peptides exhibited a superposition of predominantly unordered structure and partial contributions from beta-sheet structure. Likewise, the analysis of the amide I and amide III bands from micro-Raman and FT Raman experiments revealed mainly unordered conformations of the cyclic and of the linear peptide. NMR data demonstrated the presence of a beta-hairpin among an ensemble of largely disordered structures in the cyclic peptide. The location of the turn linking the two strands of the hairpin was assigned to the three central residues L-296, N-297, and R-298. A small population of structured species among an ensemble of predominantly random coil conformation suggests that the unliganded receptor represents a variety of structural conformers, some of which have the potential to make contacts with the ligand. We propose a mechanism of receptor activation whereby binding of the agonist to the inactive receptor state induces and stabilizes a particular structural state of the loop domain, leading to further conformational rearrangements across the transmembrane domain and signal propagating interaction with G proteins. Interaction of the Glu(301) of the receptor with Arg(8) of GnRH induces a folded configuration of the ligand. Our proposal thus suggests that conformational changes of both ligand and receptor result from this interaction.

Distribution of two splice variants of the glutamate transporter GLT-1 in the developing rat retina

The distributions of a carboxyl terminal splice variant of the glutamate transporter GLT-1, referred to as GLT-1B, and the carboxyl terminus of the originally described variant of GLT-1, referred to hereafter as GLT-1alpha, were examined using specific antisera. GLT-1B was present in the retina at very early developmental stages. Labelling was demonstrable at embryonic day 14, and strong labelling was evident by embryonic day 18. Such labelling was initially restricted to populations of cone photoreceptors, the processes of which extended through the entire thickness of the retina and appeared to make contact with the retinal ganglion cells. During postnatal development the GLT-1B-positive photoreceptor processes retracted to form the outer plexiform layer, and around postnatal day 7, GLT-1B-immunoreactive bipolar cells appeared. The pattern of labelling of bipolar cell processes within the inner plexiform layer changed during postnatal development. Two strata of strongly immunoreactive terminals were initially evident in the inner plexiform layer, but by adulthood these two bands were no longer evident and labelling was restricted to the somata and processes (but not synaptic terminals) of the bipolar cells, as well as the somata, processes, and terminals of cone photoreceptors. By contrast, GLT-1alpha appeared late in postnatal development and was restricted mainly to a population of amacrine cells, although transient labelling was also associated with punctate elements in the outer plexiform layer, which may represent photoreceptor terminals, (C) 2002 Wiley-Liss, Inc.

Adaptation of rainbow fish to lake and stream habitats

Fish occupy a range of hydrological habitats that exert different demands on locomotor performance. We examined replicate natural populations of the rainbow fishes Melanotaenia eachamensis and M. duboulayi to determine if colonization of low-velocity (lake) habitats by fish from high-velocity (stream) habitats resulted in adaptation of locomotor morphology and performance. Relative to stream conspecifics, lake fish had more posteriorly positioned first dorsal and pelvic fins, and shorter second dorsal fin bases. Habitat dimorphism observed between wild-caught fish was determined to be heritable as it was retained in M. eachamensis offspring raised in a common garden. Repeated evolution of the same heritable phenotype in independently derived populations indicated body shape divergence was a consequence of natural selection. Morphological divergence between hydrological habitats did not support a priori expectations of deeper bodies and caudal peduncles in lake fish. However, observed divergence in fin positioning was consistent with a family-wide association between habitat and morphology, and with empirical studies on other fish species. As predicted, decreased demand for sustained swimming in takes resulted in a reduction in caudal red muscle area of lake fish relative to their stream counterparts. Melanotaenia duboulayi lake fish also had slower sustained swimming speeds (U-crit) than stream conspecifics. In M. eachamensis, habitat affected U-crit of males and females differently. Specifically, females exhibited the pattern observed in M. duboulayi (lake fish had faster U-crit than stream fish), but the opposite association was observed in males (stream males had slower Ucrit than lake males). Stream M. eachamensis also exhibited a reversed pattern of sexual dimorphism in U-crit (males slower than females) relative to all other groups (males faster than females). We suggest that M. eachamensis males from streams responded to factors other than water velocity. Although replication of muscle and U,,it phenotypes across same habitat populations within and/or among species was suggestive of adaptation, the common garden experiment did not confirm a genetic basis to these associations. Kinematic studies should consider the effect of the position and base length of dorsal fins.