Phylogenetic analysis of evolutionary relationships of the planctomycete division of the domain bacteria based on amino acid sequences of elongation factor Tu

Sequences from the tuf gene coding for the elongation factor EF-Tu were amplified and sequenced from the genomic DNA of Pirellula marina and Isosphaera pallida, two species of bacteria within the order Planctomycetales. A near-complete (1140-bp) sequence was obtained from Pi. marina and a partial (759-bp) sequence was obtained for I. pallida. Alignment of the deduced Pi. marina EF-Tu amino acid sequence against reference sequences demonstrated the presence of a unique Il-amino acid sequence motif not present in any other division of the domain Bacteria. Pi. marina shared the highest percentage amino acid sequence identity with I. pallida but showed only a low percentage identity with other members of the domain Bacteria. This is consistent with the concept of the planctomycetes as a unique division of the Bacteria. Neither primary sequence comparison of EF-Tu nor phylogenetic analysis supports any close relationship between planctomycetes and the chlamydiae, which has previously been postulated on the basis of 16S rRNA. Phylogenetic analysis of aligned EF-Tu amino acid sequences performed using distance, maximum-parsimony, and maximum likelihood approaches yielded contradictory results with respect to the position of planctomycetes relative to other bacteria, It is hypothesized that long-branch attraction effects due to unequal evolutionary rates and mutational saturation effects may account for some of the contradictions.

Composition of pecan cultivars Wichita and Western Schley [Carya illinoinensis (Wangenh.) K. Koh] grown in Australia

Pecans from the cultivars Wichita and Western Schley [Carya illinoinensis (Wangenh.) K. Koch] collected over three years were analyzed for the following constituents: total lipid content; fatty acid profiles; sucrose content; protein; total dietary fiber; the minerals magnesium, calcium, potassium, sulfur, phosphorus, boron, copper, iron, manganese, sodium, zinc, and aluminum; vitamin C; and lipase; and lipoxygenase activities. Year of harvest and cultivar had little effect on the composition of the pecans. Overall, protein content was the only constituent that differed between pecans grown in Australia and those grown in the United States. This difference is probably related to differences in growing location and horticultural practices between the two countries.

Effects of phosphonate and salicylic acid treatments on anthracnose disease development and ripening of 'Kensington Pride' mango fruit

This study investigated treatment of mango (Mangifera indica L.) fruit with 2 host defence-promoting compounds for suppression of anthracnose disease (Colletotrichum gloeosporioides). Cultivar 'Kensington Pride' fruit were treated at concentrations of up to 1000 mg/L with either potassium phosphonate or salicylic acid. Applications were by various combinations of pre- and postharvest dips and vacuum infiltration. Postharvest treatments at up to 2000 mg/L salicylic acid were evaluated in a second fruiting season. Fruit were either uninoculated or inoculated with the fungal pathogen. Colour, firmness and disease-severity were assessed during shelf life at 23 degreesC. There were no significant (P>0.05) effects of potassium phosphonate or salicylic acid on anthracnose disease severity in the first season. Moreover, phosphonate or salicylic acid treatment did not significantly affect fruit colour or firmness changes. There were significant (P

Characterization of a glycine receptor domain that controls the binding and gating mechanisms of the beta-amino acid agonist, taurine

The beta -amino acid, taurine, is a full agonist of the human glycine receptor al subunit when recombinantly expressed in a mammalian (HEK293) cell line, but a partial agonist of the same receptor when expressed in Xenopus oocytes. Several residues in the Ala101-Thr112 domain have previously been identified as determinants of beta -amino acid binding and gating mechanisms in Xenopus oocyte-expressed receptors. The present study used the substituted cysteine accessibility method to investigate the role of this domain in controlling taurine-specific binding and gating mechanisms of glycine receptors recombinantly expressed in mammalian cells. Asn102 and Glu103 are identified as taurine and glycine binding sites, whereas Ala101 is eliminated as a possible binding site. The N102C mutation also abolished the antagonistic actions of taurine, indicating that this site does not discriminate between the putative agonist- and antagonist-bound conformations of beta -amino acids. The effects of mutations from Lys104-Thr112 indicate that the mechanism by which this domain controls beta -amino acid-specific binding and gating processes differs substantially depending on whether the receptor is expressed in mammalian cells or Xenopus oocytes. Thr112 is the only domain element in mammalian cell-expressed GlyRs which was demonstrated to discriminate between glycine and taurine.

Protein targeting to the plasma membrane of adult skeletal muscle fiber: An organized mosaic of functional domains

The plasma membrane of differentiated skeletal muscle fibers comprises the sarcolemma, the transverse (T) tubule network, and the neuromuscular and muscle-tendon junctions. We analyzed the organization of these domains in relation to defined surface markers, beta -dystroglycan, dystrophin, and caveolin-3, These markers were shown to exhibit highly organized arrays along the length of the fiber. Caveolin-3 and beta -dystroglycan/dystrophin showed distinct, but to some extent overlapping, labeling patterns and both markers left transverse tubule openings clear. This labeling pattern revealed microdomains over the entire plasma membrane with the exception of the neuromuscular and muscle-tendon junctions which formed distinct demarcated macrodomains. Our results suggest that the entire plasma membrane of mature muscle comprises a mosaic of T tubule domains together with sareolemmal caveolae and beta -dystroglycan domains. The domains identified with these markers were examined with respect to targeting of viral proteins and other expressed domain-specific markers, We found that each marker protein was targeted to distinct microdomains, The macrodomains were intensely labeled with all our markers. Replacing the cytoplasmic tail of the vesicular stomatitis virus glycoprotein with that of CD4 resulted in retargeting from one domain to another. The domain-specific protein distribution at the muscle cell surface may be generated by targeting pathways requiring specific sorting information but this trafficking is different from the conventional apical-basolateral division. (C) 2001 Academic Press.

Transcriptional suppression of Sox9 expression in chondrocytes by retinoic acid

SOX9 is a transcription factor that is expressed in chondrocytes and regulates expression of chondrocyte phenotype related genes. Expression of these genes is known to be suppressed by retinoic acid (RA). We, therefore, examined whether the Sox9 gene expression is regulated by RA in chondrocytes. RA treatment suppressed Sox9 mRNA expression in primary chondrocytes prepared from newborn mouse rib cartilage within 12 h and this suppression lasted at least up to 24 h. The RA suppression of Sox9 mRNA levels was dose-dependent starting at 0.5 muM with a maximum at 1 muM. Nuclear run-on assays revealed that RA reduced the rate of transcription of Sox9 gene. Finally, Western blot analysis indicated that RA suppressed SOX9 protein revels in these chondrocytes. Furthermore, overexpression of SOX9 reversed RA suppression of Col/2a1 enhancer activity. These observations indicate that RA suppresses Sox9 gene expression in chondrocytes at least in part through transcriptional events. (C) 2001 Wiley-Liss, Inc.

Amino acid residues in conodont elements

Thermally unaltered conodont elements, brachiopods. and vertebrates were analyzed with reverse phase high profile liquid chromatography to locate and quantify amino acid remnants of the original organic matrix in the fossils. No consistent similarities in amino acid content were found in conodont taxa. and criteria based on organic residues appear to have no taxonomic significance in the fossils tested from these localities. However, hydroxyproline. an amino acid that is found in the collagen molecules of animals. as well as in the glycoproteins in the cell walls and reproductive tissues of certain plants, is represented in most taxa. The organic matter retained in the impermeable crowns of conodont elements might have been derived originally from a form of collagen. Biochemical analyses. correlated with histochemical tests, demonstrate that organic matter is an integral part of the hyaline tissue of the element crown and not the result of surface contamination. Tests of a range of vertebrate and invertebrate fossil hard tissues produced similar results. The analyses indicate that hyaline tissue in the conodont element crown is not a form of vertebrate enamel. which contains no collagen. Albid tissue. with little or no organic content. is not a form of vertebrate bone or dentine, both based on collagen and low in mineral. Although these results do not help to determine the phylogenetic affinities of conodont animals, they indicate teat conodont elements do not contain hard tissues characteristic of vertebrate animals.

A histone deacetylase inhibitor, azelaic bishydroxamic acid, shows cytotoxicity on Epstein-Barr virus-transformed B-cell lines - A potential therapy for posttransplant lymphoproliferative disease

Background. Posttransplant lymphoproliferative disease (PTLD), driven by the presence of Epstein-Barr virus (EBV), is becoming an increasingly important clinical problem after solid organ transplantation. The use of immunosuppressive therapy leads to the inhibition of the cytotoxic T cells that normally control the EBV latently infected B cells. The prognosis for many patients with PTLD is poor, and the optimal treatment strategy is not well defined. Method. This study investigates the use of a histone deacetylase inhibitor, azelaic bishydroxamic acid (ABRA), for its ability to effectively kill EBV-transformed lymphoblastoid cell lines. Results. In vitro treatment of lymphoblastoid cell lines with ABRA showed that they were effectively killed by low doses of the drug (ID50 2-5 mug/ml) within 48 hr. As well as being effective against polyclonal B-cell lines, ABHA was also shown to be toxic to seven of eight clonal Burkitt's lymphoma cell lines, indicating that the drug may also be useful in the treatment of late-occurring clonal PTLD. In addition, ABHA treatment did not induce EBV replication or affect EBV latent gene expression. Conclusion. These studies suggest that ABHA effectively kills both polyclonal and clonal B-cell lines and has potential in the treatment of PTLD.

Dental erosion patterns from intrinsic acid regurgitation and vomiting

Background: The distribution of lesions from dental erosion due to intrinsic acid regurgitation and vomiting may be different from patterns of dental erosion due to extrinsic acids. To date studies have failed to validate this assumption. This study described the sites and nature of lesions from dental erosion in cases of intrinsic acid regurgitation, and compared them with the distribution of lesions occurring in age and sex matched controls, whose lesions are due to extrinsic acids. Methods: The University of Queensland tooth wear clinic patients were screened to select 30 cases, 21 self-identified bulimics and nine medically diagnosed chronic gastric acid regurgitators, and 30 controls. Epoxy resin models of the subjects' dentition were examined under stereoscopic light microscope at magnification 16 to 40. The patterns and sites of tooth wear were recorded for teeth representative of 20 tooth sites in every subject. Results: While the incisal edges of maxillary and mandibular anterior teeth of acid regurgitators were more frequently affected by erosion, incisal attrition was more common on controls' teeth. Cervical lesions were more commonly found in association with incisal attrition in the controls, and in association with incisal erosion in the cases. In 10 per cent of sites in case subjects, cervical lesions associated with incisal erosion were found on the lingual aspects of their mandibular incisors, canines and premolars. These lesions were almost exclusive to the case subjects. Conclusions: These results validate that lingual cervical lesions associated with incisal erosion on the mandibular anterior teeth are strong discriminators between tooth wear in patients with bulimia nervosa or chronic gastro-oesophageal reflux and those whose dental erosion is due to extrinsic acids.

Improvements to peroxide oxidation methods for analysing sulfur in acid sulfate soils

Improvements to peroxide oxidation methods for analysing acid sulfate soils (ASS) are introduced. The soil solution ratio has been increased to 1 : 40, titrations are performed in suspension, and the duration of the peroxide digest stage is substantially shortened. For 9 acid sulfate soils, the peroxide oxidisable sulfur value obtained using the improved method was compared with the reduced inorganic sulfur result obtained using the chromium reducible sulfur method. Their regression was highly significant, the slope of the regression line was not significantly different (P = 0.05) from unity, and the intercept not significantly different from zero. A complete sulfur budget for the improved method showed there was no loss of sulfur as has been reported for earlier peroxide oxidation techniques. When soils were very finely ground, efficient oxidation of sulfides was achieved, despite the milder digestion conditions. Highly sulfidic and organic soils were shown to be the most difficult to analyse using either the improved method or the chromium method. No single analytical method can be universally applied to all ASS, rather a suite of methods is necessary for a thorough understanding of many ASS. The improved peroxide method, in combination with the chromium method and the 4 M HCl extraction, form a sound platform for informed decision making on the management of acid sulfate soils.

Preliminary characterisation and extraction of anterior adhesive secretion in monogenean (platyhelminth) parasites

Secreted anterior adhesives, used for temporary attachment to epithelial surfaces of fishes (skin and gills) by some monogenean (platyhelminth) parasites have been partially characterised. Adhesive is composed of protein. Amino acid composition has been determined for seven monopisthocotylean monogeneans. Six of these belong to the Monocotylidae and one species, Entobdella soleae (van Beneden et Hesse, 1864) Johnston, 1929, is a member of the Capsalidae. Histochemistry shows that the adhesive does not contain polysaccharides, including acid mucins, or lipids. The adhesive before secretion and in its secreted form contains no dihydroxyphenylalanine (dopa). Secreted adhesive is highly insoluble, but has a soft consistency and is mechanically removable from glass surfaces. Generally there are high levels of glycine and alanine, low levels of tyrosine and methionine, and histidine is often absent. However, amino acid content varies between species, the biggest differences evident when the monocotylid monogeneans were compared with E. soleae. Monogenean adhesive shows similarity in amino acid profile with adhesives from starfish, limpets and barnacles. However, there are some differences in individual amino acids in the temporary adhesive secretions of, on the one hand, the monogeneans and, on the other hand, the starfish and limpets. These differences may reflect the fact that monogeneans, unlike starfish and barnacles, attach to living tissue (tissue adhesion). A method of extracting unsecreted adhesive was investigated for use in further characterisation studies on monogenean glues.

Micro-mechanical approximation to the stress field around an inclined fiber of FRCC

Matrix spalling or crushing is one of the important mechanisms of fiber-matrix interaction of fiber reinforced cementitious composites (FRCC). The fiber pullout mechanisms have been extensively studied for an aligned fiber but matrix failure is rarely investigated since it is thought not to be a major affect. However, for an inclined fiber, the matrix failure should not be neglected. Due to the complex process of matrix spalling, experimental investigation and analytical study of this mechanism are rarely found in literature. In this paper, it is assumed that the load transfer is concentrated within the short length of the inclined fiber from the exit point towards anchored end and follows the exponential law. The Mindlin formulation is employed to calculate the 3D stress field. The simulation gives much information about this field. The 3D approximation of the stress state around an inclined fiber helps to qualitatively understand the mechanism of matrix failure. Finally, a spalling criterion is proposed by which matrix spalling occurs only when the stress in a certain volume, rather than the stress at a small point, exceeds the material strength. This implies some local stress redistribution after first yield. The stress redistribution results in more energy input and higher load bearing capacity of the matrix. In accordance with this hypothesis, the evolution of matrix spalling is demonstrated. The accurate prediction of matrix spalling needs the careful determination of the parameters in this model. This is the work of further study. (C) 2002 Elsevier Science Ltd. All rights reserved.

Kainic acid induces 14-3-3 zeta expression in distinct regions of rat brain

Areas of the limbic system of adult male Wistar rats were screened for kainic-acid-induced gene expression. Polymerase-chain-reactionbased differential display identified a 147-bp cDNA fragment, which represented an mRNA that was upregulated in the entorhinal cortex and hippocampus in the kainic-acid-treated animals. The sequence was 97.8% homologous to rat 14-3-3 zeta isoform mRNA. Detailed Northern analysis revealed increased mRNA levels in the entorhinal cortex I h after kainic acid exposure and continued elevation 24 h post-injection in both the entorhinal cortex and hippocampus. Western blot analyses confirmed that the protein product of this gene was also present in increased amounts over the same time period. Immunohistochemistry and terminal transferase-mediated dUTP nick end labelling (TUNEL) detected expression of 14-3-3 protein exclusively in the entorhinal cortex and hippocampus, and only in TUNEL-positive neuronal cells. Expression of the tumor suppressor protein, p53 was also induced by kainate injection, and was co-localized with 14-3-3 zeta protein in selected cells only in the affected brain regions. The increase gene expression of 14-3-3 represents a transcription-mediated response associated with region selective neuronal damage induced by kainic acid. (C) 2002 Elsevier Science B.V. All rights reserved.