A new lipid from an Australian marine sponge, Callyspongia sp.

A specimen of the sponge Callyspongia sp. collected off the coast of New South Wales, Australia, has yielded the novel lipid (6Z,9Z,12Z,15Z)-1, 6, 9, 12, 15-octadecapenten-3-one, together with (4Z,7Z,10Z,13Z)-4, 7, 10, 13-hexadecatetraenoic acid.

Lipopolysaccharide biosynthesis genes in koala type I Chlamydia: Cloning and characterization

We showed in 1988 that there are two strains of Chlamydia psittaci which infect the koala (Phascolarctos cinereus). In order to further investigate the role of these chlamydial strains in pathogenesis, we have attempted to identify genes of koala type I strain chlamydial which are involved in the immunogenic response, Transformation of Escherichia coli with a plasmid containing a 6.3-kb fragment (pKOC-10) of C. psittaci DNA caused the appearance of a specific chlamydial lipopolysaccharide (LPS) epitope on the host strain. The smallest DNA fragment capable of inducing the expression of chlamydial LPS was an Xbal fragment, 2.4 kb in size (pKOC-5). DNA sequence analysis of the complete fragment revealed regions of high identity, at the amino acid level, to the gseA genes of C. pneomoniae, C. psittaci 6BC and C. trachomatis, and the kdtA gene of E. coli which code for transferases catalysing the addition of 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A. Two open reading frames (ORFs) of 1,314 and 501 nucleotides in size, within the 2.4-kb fragment, were evident, and mRNA species corresponding to these ORFs were detected by Northern analysis. Both ORF1 and ORF2 are required for the appearance of chlamydia-specific LPS on the surface of recombinant E. coli.

The effects of copper on the microbial community of a coral reef sponge

Marine sponges often harbour communities of symbiotic microorganisms that fulfil necessary functions for the well-being of their hosts. Microbial communities associated with the sponge Rhopaloeides odorabile were used as bioindicators far sublethal cupric ion (Cu2+) stress. A combined strategy incorporating molecular, cultivation and electron microscopy techniques was adopted to monitor changes in microbial diversity. The total density of sponge-associated bacteria and counts of the predominant cultivated symbiont (alpha -proteobacterium strain NW001) were significantly reduced in response to Cu2+ concentrations of 1.7 mug l(-1) and above after 14 days of exposure. The number of operational taxonomic units (OTUs) detected by restriction fragment length polymorphism (RFLP) decreased by 64% in sponges exposed to 223 mug l(-1) Cu2+ for 48 h and by 46% in sponges exposed to 19.4 mug l(-1) Cu2+ for 14 days. Electron microscopy was used to identify 17 predominant bacterial morphotypes, composing 47% of the total observed cells in control sponges. A reduction In the proportion of these morphotypes to 25% of observed cells was evident in sponges exposed to a Cu2+ concentration of 19.4 mug l(-1). Although the abundance of most morphotypes decreased under Cu2+ stress, three morphotypes were not reduced in numbers and a single morphotype actually increased in abundance. Bacterial numbers, as detected using fluorescence in situ hybridization (FISH), decreased significantly after 48 h exposure to 19.4 mug l(-1) Cu2+. Archaea, which are normally prolific in R. odorabile, were not detected after exposure to a Cu2+ concentration of 19.4 mug l(-1) for 14 days, indicating that many of the microorganisms associated with R. odorabile are sensitive to free copper. Sponges exposed to a Cu2+ concentration of 223 mug l(-1) became highly necrosed after 48 h and accumulated 142 +/- 18 mg kg(-1) copper, whereas sponges exposed to 19.4 mug l(-1) Cu2+ accumulated 306 +/- 15 mg kg(-1) copper after 14 days without apoptosis or mortality. Not only do sponges have potential for monitoring elevated concentrations of heavy metals but also examining changes in their microbial symbionts is a novel and sensitive bioindicator for the assessment of pollution on important microbial communities.

Guadalupian algae-sponge reefs in siliciclastic environments - the reefs at Lengwu (South China) compared with the reef at Iwaizaki (Japan)

Guadalupian reefs occur locally in Guangxi, Guizhou, Yunnan and Western Zhejiang, South China. Two types of Guadalupian reefs can be recognized, one is developed in carbonate platforms, e.g. those in the juncture areas of Guangxi, Yunnan and Guizhou; the other occurs in a littoral clastic shelf. The Lengwu reef in Western Zhejiang is a representative of the latter type, which is a major topic of this paper. Lengwu algae-sponge reef, more than one hundred meters in thickness, are composed mainly of sponges, hydrozoans, algae, bryozoans, microbes and lime mud. Reef limestones sit on the mudstone interbedded with fine sandstone of the proximal prodelta facies and are overlain by coarse clasts of the delta front sediments. Lengwu reef displays a lens-shaped relief, dipping and thinning from the reef core, which is remarkably different from the surrounding sediments, showing a protruding relief. Sponges and microbe/algae form bafflestone, bindstone and framestone of the reef core facies. Fore-reef facies is characterized by lithoclastic rudstone and bioclastic packstone. Reef limestone sequence is composed of three cycles and controlled by sea level changes and sediment influx. Such reef is unique among the Guadalupian reefs in South China, but seems similar in some aspects to lwaizaki reef limestones of south Kitakami in Japan. Algae and microbes growing around sponges to form rigid structure in Lengwu reef are a typical feature, which is distinctly different to Guadalupian reefs in a stable platform facies of Guizhou, Yunnan and Guangxi, South China.

Insulin-like growth factor 2 cDNA cloning and ontogeny of gene expression in the liver of the marsupial brushtail possum (Trichosurus vulpecula)

The cDNA sequence for insulin-like growth factor 2 (IGF-2) was determined from the liver of the marsupial brushtail possum (Trichosurus vulpecula) using reverse transcription followed by polymerase chain reaction (RT-PCR) with gene-specific primers. The 359 bp of possum sequence encompassed the mature peptide, 27 bp of the signal peptide, and 125 bp of the E-peptide. Alignment of the deduced amino acid sequence with those from other species indicated that the mature peptide was 71 amino acids in length, 4 amino acids longer than most other mammals. At both the nucleotide and amino acid levels there was a high degree of sequence identity with IGF-2 from other mammalian and nonmammalian species. Amino acid identity ranged from 94.4% with a variant form of human IGF-2 to 80.3% with zebrafinch IGF-2. Northern analysis revealed that radiolabeled possum IGF-2, cDNA hybridized to multiple transcripts in the liver of both adult possums and 150-day-old pouch young and that the overall level of expression was greater in pouch young. Semiquantitative RT-PCR with total RNA from liver samples of pouch young aged 12 to 150 days postpartum and adults confirmed that IGF-2 gene expression was two to three times more abundant in pouch young than in adults but there was no significant change in the level of expression during pouch life. Unlike other mammalian species, in which there is a decline in levels of liver IGF-2 gene expression around the time of birth, levels in the marsupial brushtail possum remain elevated for at least 150 days after birth. This suggests that the decline in liver IGF-2 expression in marsupials and eutherians occurs at a similar stage of development and may reflect a role for this growth factor during the postnatal growth and development of the marsupial, (C) 2001 Academic Press.

Cloning and function of the rat colonic epithelial K+ channel K(V)LQT1

K(V)LQT1 (K(V)LQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K+ channel in the colonic epithelium. We now report cloning and expression of K(V)LQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to K(V)LQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of K(V)LQT1 in crypt cells and surface epithelium. Expression of rK(V)LQT1 in Xenopus oocytes induced a typical delayed activated K+ current. that was further activated by increase of intracellular cAMP but not Ca2+ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K+ conductance in the colonic mucosal epithelium and inhibited whole cell K+ currents in patch-clamp experiments on isolated colonic crypts. We conclude that K(V)QT1 is forming an important component of the basolateral cAMP-activated K+ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis.

Antifungal alkyl amino alcohols from the tropical marine sponge Haliclona n. sp.

Three new amino alcohols presumably deriving from L-alanine were isolated from the tropical marine sponge Haliclona n. sp. and characterized by 2D NMR, while a fourth amino alcohol was characterized as an acetamide derivative. Relative stereochemistry was deduced from the NMR characteristics of oxazolidinone derivatives and absolute stereochemistry secured by preparation and analysis of an MPA ester. The amino alcohol fraction from Haliclona n. sp, acts as an antifungal agent and inhibits the development of larvae of the ascidian Herdmania curvata.

Advanced precursors in marine biosynthetic study. Part 2: The biosynthesis of isocyanides and isothiocyanates in the tropical marine sponge Axinyssa n.sp.

The biosynthetic origins of the isocyanide and isothiocyanate groups in 9-isocyanop upukeanane (2) and 9-isothiocyanato-pupukeanane (3) are investigated by incorporation of [C-14]-labelled advanced precursors into the sponge Axinyssa n.sp. (C) 2001 Elsevier Science Ltd. All rights reserved.

New chlorinated peptides from the tropical marine sponge Dysidea sp.

Five new chlorinated peptides (5)-(9) have been isolated from a Dysidea sp. and identified by two-dimensional NMR spectroscopy. The absolute stereochemistry of the metabolites was deduced by chemical correlation with S-(-)-4,4,4-trichloro-3-methylbutanoic acid (10) and with an alcohol (11). (C) 2001 Elsevier Science Ltd. All rights reserved.

Molecular cloning and characterization of hNP22: a gene up-regulated in human alcoholic brain

An improved differential display technique was used to search for changes in gene expression in the superior frontal cortex of alcoholics, A cDNA fragment was retrieved and cloned. Further sequence of the cDNA was determined from 5' RACE and screening of a human brain cDNA library. The gene was named hNP22 (human neuronal protein 22). The deduced protein sequence of hNP22 has an estimated molecular mass of 22.4 kDa with a putative calcium-binding site, and phosphorylation sites for casein kinase II and protein kinase C. The deduced amino acid sequence of hNP22 shares homology (from 67% to 42%) with four other proteins, SM22 alpha, calponin, myophilin and mp20. Sequence homology suggests a potential interaction of hNP22 with cytoskeletal elements. hNP22 mRNA was expressed in various brain regions but in alcoholics, greater mRNA expression occurred in the superior frontal cortex, but not in the primary motor cortex or cerebellum. The results suggest that hNP22 may have a role in alcohol-related adaptations and may mediate regulatory signal transduction pathways in neurones.

Cloning of a catalytic subunit of cAMP-dependent protein kinase from the honeybee (Apis mellifera) and its localization in the brain

In the honeybee the cAMP-dependent signal transduction cascade has been implicated in processes underlying learning and memory, The cAMP-dependent protein kinase (PKA) is the major mediator of cAMP action. To characterize the PKA system in the honeybee brain we cloned a homologue of a PKA catalytic subunit from the honeybee,The deduced amino acid sequence shows 80-94% identity with catalytic subunits of PKA from Drosophila melanogaster, Aplysia californica and mammals. The corresponding gene is predominantly expressed in the mushroom bodies, a structure that is involved in learning and memory processes. However, expression can also be found in the antennal and optic lobes,The level of expression varies within all three neuropiles.

Cloning and gastrointestinal expression of rat hephaestin: relationship to other iron transport proteins

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.