Identification of intracellular calcium oxalate crystals in Chamelaucium uncinatum (Myrtaceae)

Intracellular inclusions in the pedicel and calyx-tube tissues of Chamelaucium uncinatum Schauer ( Myrtaceae) flowers are irregular in shape. They were shown, by polarised light and scanning electron microscopy, to be birefringent 8.9-29.5 mum druse (i.e. aggregate) crystals. Energy-dispersive X-ray spectroscopy showed that these crystals were predominantly composed of calcium. Histochemical and acid-solubility tests indicated that the crystals were calcium oxalate. Raman microprobe spectroscopy was used to confirm this chemical identity. The calcium oxalate crystals were located in xylem-vessel lumens and also in parenchyma cells adjacent to vascular tissues. Thus, the crystals may function to regulate soluble calcium concentrations in C. uncinatum tissues near sites where calcium is unloaded from the xylem.

Candidatus "Scalindua brodae", spec. nov., Candidatus "Scalindua wagneri", spec. nov., two new species of anaerobic ammonium oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is both a promising process in wastewater treatment and a long overlooked microbial physiology that can contribute significantly to biological nitrogen cycling in the world's oceans. Anammox is mediated by a monophyletic group of bacteria that branches deeply in the Planctomycetales. Here we describe a new genus and species of anaerobic ammonium oxidizing planctomycetes, discovered in a wastewater treatment plant (wwtp) treating landfill leachate in Pitsea, UK. The biomass from this wwtp showed high anammox activity (5.0 +/- 0.5 nmol/mg protein/min) and produced hydrazine from hydroxylamine, one of the unique features of anammox bacteria. Eight new planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass. Four of these were affiliated to known anammox 16S rRNA gene sequences, but branched much closer to the root of the planctomycete line of descent. Fluorescence in situ hybridization (FISH) with oligonucleotide probes specific for these new sequences showed that two species (belonging to the same genus) together made up > 99% of the planctomycete population which constituted 20% of the total microbial community. The identification of these organisms as typical anammox bacteria was confirmed with electron microscopy and lipid analysis. The new species, provisionally named Candidatus Scalindua brodae and Scalindua wagneri considerably extend the biodiversity of the anammox lineage on the 16S rRNA gene level, but otherwise resemble known anammox bacteria. Simultaneously, another new species of the same genus, Candidatus Scalindua sorokinii, was detected in the water column of the Black Sea, making this genus the most widespread of all anammox bacteria described so far.

Polymerase chain reaction tests for the identification of Ross River, Kunjin and Murray Valley encephalitis virus infections in horses

Objective To develop and validate specific, sensitive and rapid diagnostic tests using RT-PCR for the detection of Ross River virus (RRV), Kunjin virus (KV) and Murray Valley encephalitis virus (MVEV) infections in horses. Methods Primer sets based on nucleotide sequence encoding the envelope glycoprotein E2 of RRV and on the nonstructural protein 5 (NS5) of KV and MVEV were designed and used in single round PCRs to test for the respective viruses in infected cell cultures and, in the case of RRV, in samples of horse blood and synovial fluid. Results The primer pairs designed for each of the three viruses amplified a product of expected size from prototype viruses that were grown in cell culture. The identity of each of the products was confirmed by nucleotide sequencing indicating that in the context used the RT-PCRs were specific. RRV was detected in serums from 8 horses for which there were clinical signs consistent with RRV infection such that an acute-phase serum sample was taken and submitted for RRV serology testing. The RRV RT-PCR was analytically sensitive in that it was estimated to detect as little as 50 TCID50 of RRV per mL of serum and was specific in that the primer pairs did not amplify other products from the 8 serum samples. The RRV primers also detected virus in three independent mosquito pools known to contain RRV by virus isolation in cell culture. Samples from horses suspected to be infected with KV and MVEV were not available. Conclusion Despite much anecdotal and serological evidence for infection of horses with RRV actual infection and associated clinical disease are infrequently confirmed. The availability of a specific and analytically sensitive RT-PCR for the detection of RRV provides additional opportunities to confirm the presence of this virus in clinical samples. The RTPCR primers for the diagnosis of KV and MVEV infections were shown to be specific for cell culture grown viruses but the further validation of these tests requires the availability of appropriate clinical samples from infected horses.

Complete genomic sequence of the Australian south-west genotype of Sindbis virus: Comparisons with other Sindbis strains and identification of a unique deletion in the 3 '-untranslated region

Our previous studies have shown that two distinct genotypes of Sindbis (SIN) virus occur in Australia. One of these, the Oriental/Australian type, circulates throughout most of the Australian continent, whereas the recently identified south-west (SW) genetic type appears to be restricted to a distinct geographic region located in the temperate south-west of Australia. We have now determined the complete nucleotide and translated amino acid sequences of a SW isolate of SIN virus (SW6562) and performed comparative analyses with other SIN viruses at the genomic level. The genome of SW6562 is 11,569 nucleotides in length, excluding the cap nucleotide and poly (A) tail. Overall this virus differs from the prototype SIN virus (strain AR339) by 23% in nucleotide sequence and 12.5% in amino acid sequence. Partial sequences of four regions of the genome of four SW isolates were determined and compared with the corresponding sequences from a number of SIN isolates from different regions of the World. These regions are the non-structural protein (nsP3), the E2 gene, the capsid gene, and the repeated sequence elements (RSE) of the 3'UTR. These comparisons revealed that the SW SIN viruses were more closely related to South African and European strains than to other Australian isolates of SIN virus. Thus the SW genotype of SIN virus may have been introduced into this region of Australia by viremic humans or migratory birds and subsequently evolved independently in the region. The sequence data also revealed that the SW genotype contains a unique deletion in the RSE of the 3'UTR region of the genome. Previous studies have shown that deletions in this region of the SIN genome can have significant effects on virus replication in mosquito and avian cells, which may explain the restricted distribution of this genotype of SIN virus.

Identification of a digalactosyl ononitol from seeds of adzuki bean (Vigna angularis)

A digalactosyl ononitol was isolated from seeds of adzuki bean (Vigna angularis [Willd.] Ohwi et Ohasi). Analysis of hydrolysis products and NMR spectroscopy established its structure as O-alpha-D-galactopyranosyl-(1-->6)-O-alpha-D-galactopyranosyl-(1-->3)-O-methyl- D-myo-inositol. (C) 2003 Elsevier Ltd. All rights reserved.

Full-scale demonstration of biological nutrient removal in a single tank SBR process

Complete biological nutrient removal (BNR) in a single tank, sequencing batch reactor (SBR) process, is demonstrated here at full-scale on a typical domestic wastewater. The unique feature of the UniFed process is the introduction of the influent into the settled sludge blanket during the settling and decant periods of the SBR operation. This achieves suitable conditions for denitrification and anaerobic phosphate release which is critical to successful biological phosphorus removal, It also achieves a selector effect, which helps in generating a compact, well settling biomass in the reactor. The results of this demonstration show that it is possible to achieve well over 90% removal of GOD, nitrogen and phosphorus in such a process. Effluent quality achieved over a six-month operating period directly after commissioning was: 29 mg/l GOD, 0.5 mg/l NH4-N, 1.5 mg/l NOx-N and 1.5 mg/l PO4-P (50%-iles of daily samples). During an 8-day, intensive sampling period, the effluent BOD5 was

Identification of a novel protein kinase mediating Akt survival signaling to the ATM protein

We identified a novel human AMP-activated protein kinase (AMPK) family member, designated ARK5, encoding 661 amino acids with an estimated molecular mass of 74 kDa. The putative amino acid sequence reveals 47, 45.8, 42.4, and 55% homology to AMPK-alpha1, AMPK-alpha2, MELK and SNARE respectively, suggesting that it is a new member of the AMPK family. It has a putative Akt phosphorylation motif at amino acids 595600, and Ser(600) was found to be phosphorylated by active Akt resulting in the activation of kinase activity toward the SAMS peptide, a consensus AMPK substrate. During nutrient starvation, ARK5 supported the survival of cells in an Akt-dependent manner. In addition, we also demonstrated that ARK5, when activated by Akt, phosphorylated the ATM protein that is mutated in the human genetic disorder ataxia-telangiectasia and also induced the phosphorylation of p53. On the basis of our current findings, we propose that a novel AMPK family member, ARK5, is the tumor cell survival factor activated by Akt and acts as an ATM kinase under the conditions of nutrient starvation.

Evidence for a sub-morphological inflammatory process in the liver in haemochromatosis

Background/Aims: The role of cytokines in hepatic injury has been examined for many liver diseases however little is known of the cytokine involvement in haemochromatosis. The aim of the current study was to examine the hepatic gene expression of potential proinflammatory and profibrogenic cytokines in haemochromatosis. Methods: Interferon-gamma, interleukin-10, transforming growth factor-beta(1) and tumor necrosis factor-alpha mRNA expression was assessed in liver tissue from 20 haemochromatosis patients, eight controls and eight chronic hepatitis C patients. To assess the immunophenotype of the inflammatory infiltrate in haemochromatosis, liver sections were subjected to immunohistochemistry using markers for macrophages (CD68, HAM56, MAC387) or T cells (CD3 and CD45RO). Results: Interferon-gamma mRNA was increased in both haemochromatosis (0.29+/-0.08%, P=0.01) and hepatitis C patients (1.02+/-0.32%, P=0.03) compared to controls (0.04+/-0.01%). Interleukin-10 mRNA was significantly decreased in both haemochromatosis and hepatitis C patients (0.01+/-0.003%, P=0.008 and 0.03+/-0.015%, P=0.02, respectively) compared to controls (0.12+/-0.01%). CD3 positive T-cell number was significantly correlated with increasing hepatic iron concentration (r=0.56, P=0.03). Conclusions: This study has demonstrated a distinct pattern of cytokine gene expression in haemochromatosis, which resembles that of inflammatory conditions such as chronic hepatitis C. These factors may play a role in the development of iron-induced hepatic fibrosis in haemochromatosis. (C) 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

Identification of residues which regulate activity of the STE20-related kinase hMINK

Activity of the STE20-related kinase hMINK was investigated. hMINK was expressed widely, though not ubiquitously, in human tissues: highest levels being found in haematopoietic tissues but also in brain, placenta, and lung. Mutagenesis revealed that T-191. and Y-193 in the substrate recognition loop of the catalytic domain were critical for kinase activity against exogenous substrates and autophosphorylation. A mutation on T-187 showed reduced enzymatic activity against exogenous substrates but retained autophosphorylationactivity. Phosphorylation was confirmed by the use of a phospho-specific T-187 antibody. hMINK activated the JNK signal transduction pathway and optimal JNK activation occurred when the C-terminus was deleted. In addition, overexpression of the C-terminal domain devoid of kinase activity also resulted in significant activation of the JNK pathway. These data suggest that hMINK requires an activation step that dissociates the C terminal, thereby freeing the catalytic domain to interact with substrates. Models for receptor-mediated activation of hMINK are discussed. (C) 2002 Elsevier Science (USA). All rights reserved.

Identification and in vivo characterization of PpaA, a regulator of photosystem formation in Rhodobacter sphaeroides

A regulatory protein, PpaA, involved in photosystem formation in the anoxygenic phototrophic proteobacterium Rhodobacter sphaeroides has been identified and characterized in vivo. Based on the phenotypes of cells expressing the ppaA gene in extra copy and on the phenotype of the ppaA null mutant, it was concluded that PpaA activates photopigment production and puc operon expression under aerobic conditions. This is in contrast to the function of the PpaA homologue from Rhodobacter capsulatus, AerR, which acts as a repressor under aerobic conditions [Dong, C., Elsen, S., Swem, L. R. & Bauer, C. E. (2002). J Bacteriol 184, 2805-2814]. The expression of the ppaA gene increases several-fold in response to a decrease in oxygen tension, suggesting that the PpaA protein is active under conditions of low or no oxygen. However, no discernible phenotype of a ppaA null mutant was observed under anaerobic conditions tested thus far. The photosystem gene repressor PpsR mediates repression of ppaA gene expression under aerobic conditions. Sequence analysis of PpaA homologues from several anoxygenic phototrophic bacteria revealed a putative corrinoid-binding domain. It is suggested that PpaA binds a corrinoid cofactor and the availability or structure of this cofactor affects PpaA activity.

Identification and comparison of aerobic and denitrifying polyphosphate-accumulating organisms

Two laboratory-scale sequencing batch reactors (SBRs) were operated for enhanced biological phosphorus removal (EBPR) in alternating anaerobic-aerobic or alternating anaerobic-anoxic modes, respectively. Polyphosphate-accumulating organisms (PAOs) were enriched in the anaerobic-aerobic SBR and denitrifying PAOs (DPAOs) were enriched in the anaerobic-aerobic SBR. Fluorescence in situ hybridization (FISH) demonstrated that the well-known PAO, Candidatus Accumulibacter phosphatis was abundant in both SBRs, and post-FISH chemical staining with 4,6-diamidino-2-phenylindol (DAPI) confirmed that they accumulated polyphosphate. When the anaerobic-anoxic SBR enriched for DPAOs was converted to anaerobic-aerobic operation, aerobic uptake of phosphorus by the resident microbial community occurred immediately. However, when the anaerobic-aerobic SBR enriched for PAOs was exposed to one cycle with anoxic rather than aerobic conditions, a 5-h lag period elapsed before phosphorus uptake proceeded. This anoxic phosphorus-uptake lag phase was not observed in the subsequent anaerobic-aerobic cycle. These results demonstrate that the PAOs that dominated the anaerobic-aerobic SBR biomass were the same organisms as the DPAOs enriched under anaerobic-anoxic conditions. (C) 2003 Wiley Periodicals, Inc.