Trichopodiella faurei n. sp. (Ciliophora,Phyllopharyngea, Cyrtophoria): Morphological Description and Phylogenetic Analyses Based on SSU rRNA and Group I Intron Sequences

ABSTRACT. A new marine cyrtophorian ciliate Trichopodiella faurei n. sp., which belongs to the order Dysteriida, family Hartmannulidae, was investigated at the morphological and molecular levels. A combination of morphological features of the organism including the oval body shape, 2–3 contractile vacuoles, 22–28 nematodesmal rods in the cytopharyngeal basket, and 31–39 somatic kineties, distinguishes it from all other known congeners. In reconstructed small subunit (SSU) rRNA phylogenies, T. faurei groups with Isochona, a representative genus of the subclass Chonotrichia. The similarity of the infraciliature between hartmannulids and several chonotrichian examples also suggests that these taxa should be closely related. A new S943 intron belonging to group IC1 was identified in the SSU rRNA gene of this species. This intron is phylogenetically related to the S891 introns previously found in the suctorians Acineta sp. and Tokophrya lemnarum, and their internal guide sequences share four nucleotides, indicating that these introns were vertically inherited from a common phyllopharyngean ancestor and that reverse splicing might have been involved in the transposition.     

Morphology and Small Subunit rDNA Gene Sequence of Pseudoamphisiella quadrinucleata n. sp. (Ciliophora,Urostylida) from the South China Sea

ABSTRACT. The urosylid genus Pseudoamphisiella was established by Song (1996) with hitherto only two known congeners. In the present work, the morphology and infraciliature of a new member, Pseudoamphisiella quadrinucleata n. sp., a form with conspicuous alveolar layer and four macronuclear nodules isolated from the coastal waters both near Hong Kong and near Guangzhou, South China were investigated using living observation and protargol silver impregnation methods. Pseudoamphisiella quadrinucleata differs from other two known forms mainly by the number of macronuclear nodules: constantly four vs. two in Pseudoamphisiella alveolata and 24–57 in Pseudoamphisiella lacazei. To support this, the sequence of the small subunit rDNA of P. quadrinucleata n. sp. showed 14 and 74 nucleotides in comparison with that of the two known congeners, respectively, which hence firmly supports the validity of the new species.     

Characterisation of inosine monophosphate dehydrogenase expression during retinal development: differences between variants and isoforms

In mammals there are two ubiquitous, catalytically indistinguishable isoforms of inosine monophosphate dehydrogenase and mutations in the type I isoform, but not type II, cause retina-specific disorders. We have characterised the spatio-temporal expression of these proteins during development of the rat retina and performed functional investigations of the recently described retinal type I variants. Inosine monophosphate dehydrogenase was present in all immature cells throughout the retina during embryonic and neonatal development. Following eye opening and cell differentiation its distribution was restricted to the photoreceptors and bipolar cells, becoming prominent in Müller cells with aging. Type II was present in early, developing retinae whilst type I was undetectable. An isoform switch occurred around P10, after which the type I variants, type Ialpha and type Igamma, were the major forms. Functional investigations indicate type Igamma has greater catalytic activity compared with other variants and isoforms. Finally, all forms of type I show an increased propensity to form intracellular macrostructures compared to type II and these structures appear to be regulated in response to changing intracellular GTP levels. Collectively these data demonstrate that (i) type I does not play a role in early retinal development, (ii) type Igamma has greater activity and (iii) there are differences between type I and type II isoforms. These observations are consistent with the aetiology of retinitis pigmentosa and raise the possibility that programmed expression of specific inosine monophosphate dehydrogenase proteins may have arisen to meet the requirements of the cellular environment.     

Docosahexaenoic acid and 17 beta-estradiol co-treatment is more effective than 17 beta-estradiol alone in maintaining bone post-ovariectomy

Bone-protective effects of combined treatment with long chain polyunsaturated fatty acids (LCPUFAs) and estrogenic compounds following ovariectomy have previously been reported. Recent evidence suggests the n-3 LCPUFA docosahexaenoic acid (DHA, 22:6n-3) is particularly bone-protective. The aim of this study was to determine whether combined treatment with DHA and estrogenic compounds has a beneficial effect on bone mass in ovariectomized (OVX) rats. Rats were randomized into 9 groups and either ovariectomized (8 groups) or sham-operated (1 group). Using a 2 x 4 factorial design approach, OVX animals received either no estrogenic compound, genistein (20 mg/kg body weight/day), daidzein, (20 mg/kg body weight/day) or 17 beta-estradiol (1 microg/day) with or without DHA (0.5 g/kg body weight/day) for 18 weeks. Bone mineral content (BMC), area (BA), and density (BMD), plasma osteocalcin and IL-6 concentrations, and red blood cell (RBC) fatty acid composition were measured. Femur BMC was significantly greater in animals treated with DHA or 17 beta-estradiol than in ovariectomized controls. Plasma carboxylated osteocalcin was significantly higher in DHA-treated animals and total osteocalcin significantly lower in 17 beta-estradiol-treated animals compared with ovariectomized controls. There were significant interactions between treatment with estrogenic compounds and DHA for femur BMC, plasma IL-6 concentration, and RBC fatty acid composition. Combined treatment with 17beta-estradiol+DHA was more effective than either treatment alone at preserving femur BMC and lowering circulating concentrations of pro-inflammatory IL-6. The percentage of n-3 LCPUFAs in RBCs was significantly greater in animals receiving 17 beta-estradiol+DHA compared with either treatment alone. There was no beneficial effect of combined DHA and phytoestrogen treatment on bone. Results from this study raise the possibility that co-treatment with 17 beta-estradiol and DHA may allow a lower dose of 17 beta-estradiol to be used to provide the same bone-protective effects as when 17 beta-estradiol is administered alone.     

Validation of the design of feeding experiments involving [(14)C]substrates used to monitor metabolic flux in higher plants

The aim of this study was to examine whether flux through the pathways of carbohydrate oxidation is accurately reflected in the pattern of ¹⁴CO₂ release from positionally labelled [¹⁴C]substrates in conventional radiolabel feeding studies. Heterotrophic cell suspension cultures of Arabidopsis thaliana were used for this work. The presence of an alkaline trap to capture metabolically generated ¹⁴CO₂ had no significant effect on the ratio of ¹⁴CO₂ release from specifically labelled [¹⁴C]substrates, or on the metabolism of [U-¹⁴C]glucose by the cells. Although the amount of ¹⁴CO₂ captured in a conventional time-course study was only about half of that released from a sample acidified at an equivalent time point, the ratios of ¹⁴CO₂ released from different positionally labelled [¹⁴C]glucose and [1-¹⁴C]gluconate were the same in untreated and acidified samples. Less than 5% of radioactivity supplied to the growth medium as [¹⁴C]bicarbonate was incorporated into acid-stable compounds, and there was no evidence for appreciable reassimilation of ¹⁴CO₂ generated intracellularly during oxidation of [1-¹⁴C]gluconate by the cells. It is concluded that the ratio of label captured from specifically labelled [¹⁴C]glucose is a valid and convenient measure of the relative rates of oxidation of the different positional carbon atoms within the supplied respiratory substrate. However, it is argued that failure to compensate for the incomplete absorption of ¹⁴CO₂ by an alkaline trap may distort estimates of respiration that rely on an absolute measure of the amount of ¹⁴CO₂ generated by metabolism.     

Characterization of glycolytic initial metabolites and enzyme activities in developing sunflower (Helianthus annuus L.) seeds

Unlike other oilseeds (e.g. Arabidopsis), developing sunflower seeds do not accumulate a lot of starch and they rely on the sucrose that comes from the mother plant to synthesise lipid precursors. Between 10 and 25 days after flowering (DAF), when sunflower seeds form and complete the main period of storage lipid synthesis, the sucrose content of seeds is relatively constant. By contrast, the glucose and fructose content falls from day 20 after flowering and it is always lower than that of sucrose, with glucose being the minor sugar at the end of the seed formation. By studying the apparent kinetic parameters and the activity of glycolytic enzymes in vitro, it is evident that all the components of the glycolytic pathway are present in the crude seed extract. However, in isolated plastids important enzymatic activities are missing, such as the glyceraldehyde-3-phosphate dehydrogenase, involved in the conversion of glyceraldehyde 3-phosphate into 1,3-biphospho-glycerate, or the enolase that converts 2-phosphoglycerate into phosphoenolpyruvate. Hence, phosphoenolpyruvate or one of its derivatives, like pyruvate and malate from the cytosol, may be the primary carbon sources for lipid biosynthesis. Accordingly, the glucose-6-P imported into the plastid is likely to be used in the pentose phosphate pathway to produce the reducing power for lipid biosynthesis in the form of NADPH. Data from crude seed extracts indicate that enolase activity increased during seed formation, from 16 days after flowering, and that this activity was well correlated with the period of storage lipid synthesis. In addition, while the presence of some glycolytic enzymes increased during lipid synthesis, others decreased, remained constant, or displayed irregular temporal behaviour.     

Pan-European regional-scale modelling of water and N efficiencies of rapeseed cultivation for biodiesel production

The energy produced from the investment in biofuel crops needs to account for the environmental impacts on soil, water, climate change and ecosystem services. A regionalized approach is needed to evaluate the environmental costs of large-scale biofuel production. We present a regional pan-European simulation of rapeseed ( Brassica napus ) cultivation. Rapeseed is the European Union's dominant biofuel crop with a share of about 80% of the feedstock. To improve the assessment of the environmental impact of this biodiesel production, we performed a pan-European simulation of rapeseed cultivation at a 10 × 10 km scale with Environmental Policy Integrated Climate (EPIC). The model runs with a daily time step and model input consists of spatialized meteorological measurements, and topographic, soil, land use, and farm management practices data and information. Default EPIC model parameters were calibrated based on literature. Modelled rapeseed yields were satisfactory compared with yields at regional level reported for 151 regions obtained for the period from 1995 to 2003 for 27 European Union member countries, along with consistent modelled and reported yield responses to precipitation, radiation and vapour pressure deficit at regional level. The model is currently set up so that plant nutrient stress is not occurring. Total fertilizer consumption at country level was compared with IFA/FAO data. This approach allows us to evaluate environmental pressures and efficiencies arising from and associated with rapeseed cultivation to further complete the environmental balance of biofuel production and consumption.     

Functional Rescue of Mutant Human Cystathionine ��-Synthase by Manipulation of Hsp26 and Hsp70 Levels in Saccharomyces cerevisiae

Many human diseases are caused by missense substitutions that result in misfolded proteins that lack biological function. Here we express a mutant form of the human cystathionine β-synthase protein, I278T, in Saccharomyces cerevisiae and show that it is possible to dramatically restore protein stability and enzymatic function by manipulation of the cellular chaperone environment. We demonstrate that Hsp70 and Hsp26 bind specifically to I278T but that these chaperones have opposite biological effects. Ethanol treatment induces Hsp70 and causes increased activity and steady-state levels of I278T. Deletion of the SSA2 gene, which encodes a cytoplasmic isoform of Hsp70, eliminates the ability of ethanol to restore function, indicating that Hsp70 plays a positive role in proper I278T folding. In contrast, deletion of HSP26 results in increased I278T protein and activity, whereas overexpression of Hsp26 results in reduced I278T protein. The Hsp26-I278T complex is degraded via a ubiquitin/proteosome-dependent mechanism. Based on these results we propose a novel model in which the ratio of Hsp70 and Hsp26 determines whether misfolded proteins will either be refolded or degraded.Cells have evolved quality control systems for misfolded proteins, consisting of molecular chaperones (heat shock proteins) and proteases. These molecules help prevent misfolding and aggregation by either promoting refolding or by degrading misfolded protein molecules (1). In eukaryotic cells, the Hsp70 system plays a critical role in mediating protein folding. Hsp70 protein interacts with misfolded polypeptides along with co-chaperones and promotes refolding by repeated cycles of binding and release requiring the hydrolysis of ATP (2). Small heat shock proteins (sHsp)² are small molecular weight chaperones that bind non-native proteins in an oligomeric complex and whose function is poorly understood (3). In mammalian cells, the sHsp family includes the α-crystallins, whose orthologue in Saccharomyces cerevisiae is Hsp26. Studies suggest that Hsp26 binding to misfolded protein aggregates is a prerequisite for effective disaggregation and refolding by Hsp70 and Hsp104 (4, 5).Misfolded proteins can result from missense substitutions such as those found in a variety of recessive genetic diseases, including cystathionine β-synthase (CBS) deficiency. CBS is a key enzyme in the trans-sulfuration pathway that converts homocysteine to cysteine (6). Individuals with CBS deficiency have extremely elevated levels of plasma total homocysteine, resulting in a variety of symptoms, including dislocated lenses, osteoporosis, mental retardation, and a greatly increased risk of thrombosis (7). Approximately 80% of the mutations found in CBS-deficient patients are point mutations that are predicted to cause missense substitutions in the CBS protein (8). The most common mutation found in CBS-deficient patients, an isoleucine to threonine substitution at amino acid position 278 (I278T), has been observed in nearly one-quarter of all CBS-deficient patients. Based on the crystal structure of the catalytic core of CBS, this mutation is located in a β-sheet more than 10 Å distant from the catalytic pyridoxal phosphate and does not directly affect the catalytic binding pocket or the dimer interface (9).Previously, our lab has developed a yeast bioassay for human CBS in which yeast expressing functional human CBS can grow in media lacking cysteine, whereas yeast expressing mutant CBS cannot (10). We have used this assay to characterize the functional effects of many different CBS missense alleles, including I278T (7, 11). However, an unexpected finding was that it was possible to restore function to I278T and a number of other CBS missense mutations by either truncation or the addition of a second missense mutation in the C-terminal regulatory domain (12, 13). The ability to restore function by a cis-acting second mutation suggested to us that it might be possible to restore function in trans via either interaction of mutant CBS with a small molecule (i.e. drug) or a mutation in another yeast gene. In a previous study, we found that small osmolyte chemical chaperones could restore function to mutant CBS presumably by directly stabilizing the mutant CBS protein (14).In this study we report on the surprising finding that exposure of yeast to ethanol can restore function of I278T CBS by altering the ratio of the molecular chaperones Hsp26 and Hsp70. We demonstrate Hsp70 binding promotes I278T folding and activity, whereas Hsp26 binding promotes I278T degradation via the proteosome. By manipulating the levels of Hsp26 and Hsp70, we are able to show that I278T CBS protein can have enzymatic activity restored to near wild-type levels of activity. Our findings suggest a novel function for sHsps.     

Phylogenetic analyses suggest that Psammomitra (Ciliophora,Urostylida) should represent an urostylid family,based on small subunit rRNA and alpha‐tubulin gene sequence information

The morphologically unique ciliate Psammomitra has long been considered as a systematically uncertain stichotrich. This is mainly because of its highly specialized morphology and a lack of either detailed information concerning its ontogenesis, or molecular data. Based on the small subunit rRNA (SSrRNA) gene and alpha‐tubulin gene sequences, we re‐evaluated the phylogenetic position of Psammomitra retractilis using multiple algorithms. Phylogenetic trees inferred from the SSrRNA gene sequences representing a total of 53 spirotrichs demonstrated the closest relationship of Psammomitra was with Holosticha‐like taxa, with strong support, which clearly suggested that Psammomitra should be placed into the order Urostylida although it branched at a rather deep level, and is likely to be closely related to Holostichidae. With consideration to molecular evidence and morphological characters, Psammomitra should be a clearly outlined taxon at about the rank of family, i.e. Psammomitridae stat. nov. , within the order Urostylida. The improved diagnosis for this family is as follows: Urostylida possessing extremely contractile, elongated body which consists of three parts: head, trunk, and slender tail; midventral complex composed of midventral pairs only and restricted to about anterior 1/3 of ventral surface; frontal, frontoterminal, and transverse cirri present; one left and one right marginal rows which commence near proximal end of adoral zone and extend to near rear body end.     

Molecular characterization of a fungal gene paralogue of the penicillin penDE gene of Penicillium chrysogenum

Background  

Penicillium chrysogenum converts isopenicillin N (IPN) into hydrophobic penicillins by means of the peroxisomal IPN acyltransferase (IAT), which is encoded by the penDE gene. In silico analysis of the P. chrysogenum genome revealed the presence of a gene, Pc13g09140, initially described as paralogue of the IAT-encoding penDE gene. We have termed this gene ial because it encodes a protein with high similarity to IAT (IAL for IAT-Like). We have conducted an investigation to characterize the ial gene and to determine the role of the IAL protein in the penicillin biosynthetic pathway.