Sponge halogenated natural products found at parts-per-million levels in marine mammals

Several unknown, abundant brominated compounds (BCs) were recently detected in the blubber of dolphins and other marine mammals from Queensland (northeast Australia). The BC were interpreted as potential natural products due to the lack of anthropogenic sources for these compounds. This study investigated whether some of the BCs accumulated by diverse marine mammal species are identical with natural BCs previously isolated from sponges (Dysidea sp.) living in the same habitat. Isolates from sponges and mollusks (Asteronotus cespitosus) were compared with the signals detected in the mammals' tissue. Mass spectra and gas chromatography retention times on four different capillary columns of the isolates from sponges and mammals were identical in all respects. This proves that the chemical name of the compound previously labeled BC-2 is 4,6-dibromo-2-(2'-dibromo)phenoxyanisole and that the chemical name of BC-11 is 3,5-dibromo-2-(3',5'-dibromo-2'-methoxy)phenoxyanisole. Using a quantitative reference solution of BC-2, we established that the concentrations of the brominated metabolies found in the marine mammals are frequently >1 mg/kg. The highest concentration (3.8 mg/kg), found in a sample of pygmy sperm whale (Kogia breviceps), indicates that BC-2 is a bioaccumulative, natural organohalogen compound. This is supported by the concentrations of the BCs in our samples being equal to the highest concentrations of anthropogenic BCs in any environmental sample. The quantitative determination of BC-2 in blubber of marine mammals from Africa and the Antarctic suggests that BC-2 is wide-spread. These results are direct proof that marine biota can produce persistent organic chemicals that accumulate to substantial concentrations in higher trophic organisms.

Investigations into the PCDD contamination of topsoil, river sediments and kaolinite clay in Queensland, Australia

Recent findings of elevated PCDDs from an unknown source in the coastal marine environment of Queensland, Australia has instigated further investigations into the distribution of, and environments associated with the PCDD contamination. This study presents data for OCDD concentrations in the coastal, mountainous and inland environment of Queensland. Additionally, full 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and dibenzofuran (PCDD/F) profiles from different land-use types and environments in the coastal region were analysed. Distinct east-west gradients were detected in topsoil collected from various bushland regions with elevated OCDD concentrations confined to the coastal region. However, PCDD/F results from topsoil and river sediments collected in the Queensland coastal region suggest that elevated OCDD concentrations cannot be attributed to any of the environments, land-use or industry types investigated. PCDD/F congener profiles from select samples were remarkably similar to those previously described in marine sediments collected along the entire Queensland coastline. In addition, kaolinite clay samples from Queensland exhibited elevated OCDD concentrations, and PCDD/F profiles in these samples were similar to those detected in kaolinite clays elsewhere. Natural formation processes have been hypothesised as the source of elevated PCDDs in Queensland and other locations, where similar PCDD/F profiles and the general lack of anthropogenic sources are evident. This study presents additional data supporting this hypothesis and provides further information that may assist in the identification of the processes involved in the natural formation of PCDDs. (C) 2002 Elsevier Science Ltd. All rights reserved.

Discovery and structures of the cyclotides: novel macrocyclic peptides from plants

Circular disulfide-rich polypeptides were unknown a decade ago but over recent years a large family of such molecules has been discovered, which we now refer to as the cyclotides. They are typically about 30 amino acids in size, contain an N- to C-cyclised backbone and incorporate three disulfide bonds arranged in a cystine knot motif. In this motif, an embedded ring in the structure formed by two disulfide bonds and their connecting backbone segments is penetrated by the third disulfide bond. The combination of this knotted and strongly braced structure with a circular backbone renders the cyclotides impervious to enzymatic breakdown and makes them exceptionally stable. This article describes the discovery of the cyclotides in plants from the Rubiaceae and Violaceae families, their chemical synthesis, folding, structural characterisation, and biosynthetic origin. The cyclotides have a diverse range of biological applications, ranging from uterotonic action, to anti-HIV and neurotensin antagonism. Certain plants from which they are derived have a history of uses in native medicine, with activity being observed after oral ingestion of a tea made from the plants. This suggests the possibility that the cyclotides may be orally bioavailable. They therefore have a range of potential applications as a stable peptide framework.

Predation of brown tree snakes (Boiga irregularis) in Australia

We conducted a literature review to address the potential for using a native, vertebrate predator of brown tree snakes (Boiga irregularis) as a biological control method on Guam. Both actual and potential predators were included in our review. We located two actual predators (red-bellied black snakes (Pseudechis porphyriacus) and cane toads (Bufo marinus)) and 55 potential predators of brown tree snakes. However, none of the native predators of brown tree snakes appear likely candidates as a biological control method on Guam due to their lack of selectivity in their feeding habits and unknown aspects of their natural history. (C) 2002 Elsevier Science Ltd. All rights reserved.

Tree yield and fruit minerals concentrations influence 'Hass' avocado fruit quality

To investigate the variation in quality of 'Hass' avocado fruit within an orchard, fruit were harvested at commercial maturity from 15 'Hass' trees of similar appearance, growing in three adjacent rows on the same soil type, and receiving similar management. Fruit were harvested at commercial maturity, and either ripened at 22 degreesC or stored at 2 or 7 degreesC for 3 or 5 weeks and then ripened. Significant positive correlations (based, on the mean for each datum tree) were noted between fruit flesh calcium (Ca) and magnesium (Mg) concentrations and the (Ca + Mg)/potassium ratio, and the number of days for the fruit to reach the eating ripe stage (DTR). Negative correlations were also observed between these minerals and anthracnose and mesocarp discolouration (MD) severity. Negative correlations were observed between fruit potassium (K) and phosphorus (P) concentrations and DTR. Fruit from trees with high fruit yield were generally smaller, with lower anthracnose and MD severity, ripened more slowly, and had higher flesh Ca concentrations. It is likely that cultural practices that maintain moderate to high fruit yield and reduce variation in yield will improve avocado fruit quality and reduce variability in quality. Since the main differences between adjacent trees in this trial were the seedling rootstocks of unknown origin, it is suggested that rootstocks can have a significant impact on avocado yield and fruit quality. (C) 2002 Elsevier Science B.V. All rights reserved.

Amphitheater staging: In-the-round or to the front (and what about asides)?

When the existence in Utrecht of the DeWitt/Van Buchel drawing of the Swan Theater became known in 1888, William Poel was already seven years into his inquiries into the best means of staging the public amphitheater and great hall plays of the English Renaissance.1 I mention this to emphasize that the discourse came before the fact: the belief that Shakespeare's plays are best staged as it was then imprecisely imagined they had once been staged-simply, without elaborate settings or time-consuming scene changes, with direct actor-audience address, "in-the-round"-had as much to do with reactions against the late nineteenth-century stage's pictorialism, with its set-changing interruptions and cut-and-paste revisions to Shakespeare's texts, as it had to do with presenting the Bard "authentically." Some of the confusion caused by the uneasy mixing of historical scholarship and the assumptions and practices of our own contemporary theater profession can be glimpsed in the phenomenon of modern in-the-round staging, often claimed as a more "authentic" approach to Shakespeare in performance, but one which is then modified to make possible post-Stanislavsky acting methods and to satisfy modern audience expectations.

Angiotensinogen genotype, plasma protein and mRNA concentration in isolated systolic hypertension

Isolated systolic hypertension (ISH) occurs predominantly in the elderly, with a considerable morbidity and mortality. Its etiology is unknown but is likely to involve a significant genetic component. The aim of this study was to examine the angiotensinogen gene in ISH. The M235T and G(- 6)A polymorphisms were genotyped by polymerase chain reaction (PCR) in 86 ISH patients and 120 normotensive controls. Plasma angiotensinogen concentration was determined in 198 subjects by an indirect radioimmunoassay technique. Angiotensinogen mRNA concentration was determined by quantitative competitive reverse transcription (RT)-PCR in subcutaneous adipose tissue from a subset of these patients (n = 8) and controls (n = 6). Both the M235T (p = 0.0015) and G(- 6)A (p = 0.029) polymorphisms were associated with ISH. Plasma angiotensinogen concentration was higher in patients than controls (p < 0.0001), but was not associated with genotype. Angiotensinogen mRNA concentration in adipose tissue from ISH subjects was significantly lower than in adipose tissue from normotensive subjects (p = 0.033). The association of angiotensinogen gene variants with ISH and the elevation of plasma angiotensinogen concentration in these patients suggests a role of the angiotensinogen gene in this form of hypertension. Angiotensinogen gene expression may be altered in ISH, but this requires further examination.

Association of hypertension and hypokalemia with Cushing's syndrome caused by ectopic ACTH secretion: a series of 58 cases.

Cushing's syndrome is associated with hypertension in approximately 80% of cases. Hypertension contributes to the marked increased mortality risk of past or current Cushing's syndrome, largely because of increased cardiovascular risk. Observation of the pathophysiological effect of chronically elevated ACTH and cortisol values in patients with ectopic ACTH secretion complements the available data from acute studies of the effects of ACTH and glucocorticoid infusions in normal volunteers. In a retrospective case review, we identified 58 patients with Cushing's syndrome caused by ectopic ACTH secretion, who were treated at the National Institutes of Health between 1983-1997. The diagnosis of an ectopic ACTH cause was confirmed by inferior petrosal sinus sampling and/or pathologic examination of tumor. The commonest causes were bronchial carcinoid (40%) and thymic carcinoid (10%), but 18 of 58 (31%) patients had an unknown source of ectopic ACTH. Hypertension (systolic blood pressure >140 mmHg and/or diastolic blood pressure >90 mmHg in adults) was noted in 45 of 58 (78%) ectopic Cushing's patients, a prevalence similar to that noted in other endogenous Cushing's syndrome etiologies. Hypertension was severe, deemed to require 3 or more drugs by the treating physicians, in 26 of 58 (45%) patients. Hypokalemia was much more prevalent than in patients with other causes of Cushing's syndrome, affecting 33 of 58 (57%) patients. The range of plasma ACTH (17-1557 pg/mL, normal

Conformationally constrained macrocycles that mimic tripeptide beta-strands in water and aprotic solvents

The beta-strand conformation is unknown for short peptides in aqueous solution, yet it is a fundamental building block in proteins and the crucial recognition motif for proteolytic enzymes that enable formation and turnover of all proteins. To create a generalized scaffold as a peptidomimetic that is preorganized in a beta-strand, we individually synthesized a series of 15-22-membered macrocyclic analogues of tripeptides and analyzed their structures. Each cycle is highly constrained by two trans amide bonds and a planar aromatic ring with a short nonpeptidic linker between them. A measure of this ring strain is the restricted rotation of the component tyrosinyl aromatic ring (DeltaG(rot) 76.7 kJ mol(-1) (16-membered ring), 46.1 kJ mol(-1) (17-membered ring)) evidenced by variable temperature proton NMR spectra (DMF-d(7), 200-400 K). Unusually large amide coupling constants ((3)J(NH-CHalpha) 9-10 Hz) corresponding to large dihedral angles were detected in both protic and aprotic solvents for these macrocycles, consistent with a high degree of structure in solution. The temperature dependence of all amide NH chemical shifts (Deltadelta/T7-12 ppb/deg) precluded the presence of transannular hydrogen bonds that define alternative turn structures. Whereas similar sized conventional cyclic peptides usually exist in solution as an equilibrium mixture of multiple conformers, these macrocycles adopt a well-defined beta-strand structure even in water as revealed by 2-D NMR spectral data and by a structure calculation for the smallest (15-membered) and most constrained macrocycle. Macrocycles that are sufficiently constrained to exclusively adopt a beta-strand-mimicking structure in water may be useful pre-organized and generic templates for the design of compounds that interfere with beta-strand recognition in biology.

Inhibitors of COP-mediated Transport and Cholera Toxin Action Inhibit Simian Virus 40 Infection

Simian virus 40 (SV40) is a nonenveloped virus that has been shown to pass from surface caveolae to the endoplasmic reticulum in an apparently novel infectious entry pathway. We now show that the initial entry step is blocked by brefeldin A and by incubation at 20degreesC. Subsequent to the entry step, the virus reaches a domain of the rough endoplasmic reticulum by an unknown pathway. This intracellular trafficking pathway is also brefeldin A sensitive. Infection is strongly inhibited by expression of GTP-restricted ADP-ribosylation factor 1 (Arf1) and Sar1 mutants and by microinjection of antibodies to betaCOP. In addition, we demonstrate a potent inhibition of SV40 infection by the dipeptide N-benzoyl-oxycarbonyl-Gly-Phe-amide, which also inhibits late events in cholera toxin action. Our results identify novel inhibitors of SV40 infection and show that SV40 requires COPI- and COPII-dependent transport steps for successful infection.

Solution Structures of the cis- and trans-Pro30 Isomers of a Novel 38-Residue Toxin from the Venom of Hadronyche Infensa sp. that Contains a Cystine-Knot Motif within Its Four Disulfide Bonds

The primary sequence and three-dimensional structure of a novel peptide toxin isolated from the Australian funnel-web spider Hadronyche infensa sp. is reported. ACTX-HI:OB4219 contains 38 amino acids, including eight-cysteine residues that form four disulfide bonds. The connectivities of these disulfide bonds were previously unknown but have been unambiguously determined in this study. Three of these disulfide bonds are arranged in an inhibitor cystine-knot (ICK) motif, which is observed in a range of other disulfide-rich peptide toxins. The motif incorporates an embedded ring in the structure formed by two of the disulfides and their connecting backbone segments penetrated by a third disulfide bond. Using NMR spectroscopy, we determined that despite the isolation of a single native homologous product by RP-HPLC, ACTX-HI:OB4219 possesses two equally populated conformers in solution. These two conformers were determined to arise from cis/trans isomerization of the bond preceding Pro30. Full assignment of the NMR spectra for both conformers allowed for the calculation of their structures, revealing, the presence of a triple-stranded antiparallel sheet consistent with the inhibitor cystine-knot (ICK) motif.

pido, a non-long terminal repeat retrotransposon of the chicken repeat 1 family from the genome of the Oriental blood fluke, Schistosoma japonicum

A newly described non-long terminal repeat (non-LTR) retrotransposon element was isolated from the genome of the Oriental schistosome, Schistosoma japonicum. At least 1000 partial copies of the element, which was named pido, were dispersed throughout the genome of S. japonicum. As is usual with non-LTR retrotransposons, it is expected that many pido elements will be 5'-truncated. A consensus sequence of 3564 bp of the truncated pido element was assembled from several genomic fragments that contained pido-hybridizing sequences. The sequence encoded part of the first open reading frame (ORF), the entire second ORF and, at its 3'-terminus, a tandemly repetitive, A-rich (TA(6)TA(5)TA(8)) tail, The ORF1 of pido encoded a nucleic acid binding protein and ORF2 encoded a retroviral-like polyprotein that included apurinic/apyrimidinic endonuclease (EN) and reverse transcriptase (RT) domains, in that order. Based on its sequence and structure, and phylogenetic analyses of both the RT and EN domains, pido belongs to the chicken repeat 1 (CR1)-like lineage of elements known from the chicken, turtle, puffer fish, mosquitoes and other taxa. pido shared equal similarity with CRI from chicken, an uncharacterized retrotransposon from Caenorhabditis elegans and SR1 (a non-LTR retrotransposon) from the related blood fluke Schistosoma mansoni; the level of similarity between pido and SR1 indicated that these two schistosome retrotransposons were related but not orthologous. The findings indicate that schistosomes have been colonized by at least two discrete CRI-like elements. Whereas pido did not appear to have a tight target site specificity, at least one copy of pido has inserted into the 3'-untranslated region of a protein-encoding gene (GeriBank AW736757) of as yet unknown identity. mRNA encoding the RT of pido was detected by reverse transcription-polymerase chain reaction in the egg, miracidium. and adult developmental stages of S. japonicum, indicating that the RT domain was transcribed and suggesting that pido was replicating actively and mobile within the S. japonicum genome. (C) 2002 Elsevier Science B.V. All rights reserved.

Progressive Age-Related Changes Similar to Age-Related Macular Degeneration in a Transgenic Mouse Model

Age-related macular degeneration (AMD) is the major cause of blindness in the developed world. its pathomechanism is unknown and its late onset, complex genetics and strong environmental components have all hampered investigations. Here we demonstrate the development of an animal model for AMD that reproduces features associated with geographic atrophy, a transgenic mouse line (mcd/mcd) expressing a mutated form of cathepsin D that is enzymatically inactive thus impairing processing of phagocytosed photoreceptor outer segments in the retinal pigment epithelial (RPE) cells. Pigmentary changes indicating RPE cell atrophy and a decreased response to flash electroretinograms were observed in 11- to 12-month-old mcd/mcd mice. Histological studies showed RPE cell proliferation, photoreceptor degeneration, shortening of photoreceptor outer segments, and accumulation of immunoreactive photoreceptor breakdown products in the RPE cells. An accelerated photoreceptor cell death was detected in 12-month-old mcd/mcd mice. Transmission electron microscopy demonstrated presence of basal laminar and linear deposits that are considered to be the hallmarks of AMD. Small hard drusen associated with human age-related maculopathy were absent in the mcd/mcd mouse model at the ages analyzed. in summary, this model presents several features of AMD, thus providing a valuable tool for investigating the underlying biological processes and pathomechanism of AMD.

The pyramidal cell of the sensorimotor cortex of the macaque monkey: Phenotypic variation

Recent studies have revealed striking differences in pyramidal cell structure among cortical regions involved in the processing of different functional modalities. For example, cells involved in visual processing show systematic variation, increasing in morphological complexity with rostral progression from V1 through extrastriate areas. Differences have also been identified between pyramidal cells in somatosensory, motor and prefrontal cortex, but the extent to which the pyramidal cell phenotype may vary between these functionally related cortical regions remains unknown. In the present study we investigated the structure of layer III pyramidal cells in somatosensory and motor areas 3b, 4, 5, 6 and 7b of the macaque monkey. Cells were intracellularly injected in fixed, flat-mounted cortical slices and analysed for morphometric parameters. The size of the basal dendritic arbours, the number of their branches and their spine density were found to vary systematically between areas. Namely, we found a trend for increasing complexity in dendritic arbour structure through areas 3b, 5 and 7b. A similar trend occurred through areas 4 and 6. The differences in arbour structure may determine the number of inputs received by neurons and may thus be an important factor in determining function at the cellular and systems level.

Mechanisms for the inhibition of amiloride-sensitive Na+ absorption by extracellular nucleotides in mouse trachea

Purinergic stimulation of airway epithelial cells induces Cl- secretion and modulates Na+ absorption by an unknown mechanism. To gain insight into this mechanism, we used a perfused micro-Ussing chamber to assess transepithelial voltage (V-te) and amiloride-sensitive short-circuit current (Isc-Amil) in mouse trachea. Exposure to apical ATP or UTP (each 100 mumol/l) caused a large initial increase in lumen negative V-te and I-sc corresponding to a transient Cl- secretion, while basolateral application of ATP/UTP induced only a small secretory response. Luminal, but not basolateral, application of nucleotides was followed by a sustained and reversible inhibition of Isc-Amil that was independent of extracellular Ca2+ or activation of protein kinase C and was not induced by carbachol (100 mumol/l) or the Ca2+ ionophore ionomycin (1 mumol/l). Removal of extracellular Cl- or exposure to 200 muM DIDS reduced UTP-mediated inhibition of Isc-Amil Substantially. The phospholipase inhibitor U73122 (10 mumol/l) and pertussis toxin (PTX 200 ng/ml) both attenuated UTP-induced Cl- secretion and inhibition of Isc-Amil. Taken together, these data imply a contribution of Cl- conductance and PTX-sensitive G proteins to nucleotide-dependent inhibition of the amiloride-sensitive Na+ current in the mouse trachea.