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.     

Proteasome inhibition: an early or late event in nitric oxide-induced neuronal death?

Nitric oxide (NO), ubiquitously expressed in the central nervous system, has been perceived to be a potential neuromodulator. Employing cultured murine primary cortical neurons, NO resulted in an inhibition of the ubiquitin-proteasome system (UPS) with a dose- and time-dependent decrease in cell viability. This is consistent with a previous study that reported a dysfunction of UPS with consequential apoptotic death in macrophage cell with NO treatment. However, it cannot be unclear if the drop in UPS efficiency is directly imposed on by NO. Therefore by using microarray analysis, our study revealed an early down-regulation or non-significant differential expression of genes encoding UPS proteins in NOC-18 (NO donor)-treated neurons as compared to an observed elevation of corresponding gene expression genes in lactacystin (classical proteasome inhibitor)-treated neurons (conducted earlier). Furthermore, time-course analysis of proteasome activity in NOC-18-treated neurons demonstrated a late onset of reduction. This is intriguing as it is well established that in an exclusive proteasome dysfunction-induced cell death, a compensatory feedback mechanism will be activated with an initial and concerted up-regulation of genes encoding proteins involved in UPS as seen when neurons were treated with lactacystin. Thus, it is highly suggestive that NO-triggered neuronal death takes on a different signaling cascade from that of a classical proteasome inhibitor, and that the late reduction of proteasome activity is a downstream event following the activation of apoptotic cellular signaling cascade. In intracellular condition, the proteasome is not NO preferred primary target responsible for the trigger of the cell death machinery. In conclusion, we presented novel findings that shed light into NO-induced cell death signaling cascade, which would be important in understanding the pathogenesis of neurodegenerative disorders such as Parkinson's disease.     

Refining siRNA in vivo transfection: silencing SPHK1 reveals its key role in C5a-induced inflammation in vivo

The transfection of siRNA in vivo is essential for the study of gene functions, target validation, and for gene therapy. However, the successful delivery of siRNA in whole organisms is still very difficult to achieve. A high-pressure delivery technique, called the "hydrodynamics" method, has been used for siRNA transfection in mice. However, it is a method based on a high-speed and high-volume of i.v. injection, which makes it very difficult to implement in vivo, due to vascular breakage. Here, we systematically investigated ways to optimize the siRNA delivery, in order to avoid strong side effects, while achieving a high-efficiency siRNA-gene knockdown. We show here that the amount of siRNA delivered is crucial, as using too little or too much siRNA minimizes the knockdown effect. We demonstrate that by carefully identifying an optimal-minimal volume, and an optimal amount of siRNA, we achieve a high knockdown effect, with a 100% survival rate. We have previously shown that SphK1 plays a key role in anaphylatoxin (C5a) signaling in neutrophils and macrophages. Our approach, optimizing the dosage of siRNA, allowed us to successfully silence our target gene-product (SphK1) in vivo, and enabled us to validate SphK1 as a key player in our in vivo model of C5a-induced acute peritonitis and systemic inflammation including multi-organ damage, demonstrating that this improved siRNA-silencing method not only allowed us to identify SphK1 as a key therapeutic target, but brings us a step closer to the usage of siRNA for therapeutic intervention.     

Altered redox status accompanies progression to metastatic human bladder cancer

The role of reactive oxygen species (ROS) in bladder cancer progression remains an unexplored field. Expression levels of enzymes regulating ROS levels are often altered in cancer. A search of publicly available microarray data reveals that expression of mitochondrial manganese superoxide dismutase (Sod2), responsible for the conversion of superoxide (O(2)(-)) to hydrogen peroxide (H(2)O(2)), is consistently increased in high-grade and advanced-stage bladder tumors. We aimed to identify the role of Sod2 expression and ROS in bladder cancer. Using an in vitro human bladder tumor model we monitored the redox state of both nonmetastatic (253J) and highly metastatic (253J B-V) bladder tumor cell lines. 253J B-V cells displayed significantly higher Sod2 protein and activity levels compared to their parental 253J cell line. The increase in Sod2 expression was accompanied by a significant decrease in catalase activity, resulting in a net increase in H(2)O(2) production in the 253J B-V cell line. Expression of the prometastatic and proangiogenic factors matrix metalloproteinase 9 (MMP-9) and vascular endothelial-derived growth factor (VEGF), respectively, was upregulated in the metastatic line. Expression of both MMP-9 and VEGF was shown to be H(2)O(2)-dependent, as removal of H(2)O(2) by overexpression of catalase attenuated their expression. Similarly, expression of catalase effectively reduced the clonogenic activity of 253J B-V cells. These findings indicate that metastatic bladder cancer cells display an altered antioxidant expression profile, resulting in a net increase in ROS production, which leads to the induction of redox-sensitive protumorigenic and prometastatic genes such as VEGF and MMP-9.     

Synchronization and resynchronization of inseminations in lactating dairy cows with the CIDR insert and the Ovsynch protocol

Pregnancy per artificial insemination (AI) was evaluated in dairy cows (Bos taurus) subjected to synchronization and resynchronization for timed AI (TAI). Cows (n = 718) received prostaglandin F_2α (PGF) on Days –38 and –24 (Days 39 and 53 postpartum), gonadotropin-releasing hormone (GnRH) on Day –10, PGF on Day –3, and GnRH and TAI on Day 0. Between Days –10 and –3, cows received a progesterone intravaginal insert (CIDR group) or no CIDR (Control group). Between Days 14 and 23, cows received a CIDR (Resynch CIDR group) or no CIDR (Resynch control group), GnRH on Day 23, with pregnancy diagnosis on Day 30. Cows in estrus (between Days 0 and 30) were re-inseminated at detected estrus (RIDE). Nonpregnant cows received PGF on Day 30 and GnRH and TAI on Day 33. Plasma progesterone was determined to be low or high on Days –24 and –10. Pregnancy rates were evaluated 30 and 55 d after AI. The CIDR insert included in the Presynch-Ovsynch protocol did not increase overall pregnancy per AI for first service (36.1% and 33.6% for CIDR; 34.1% and 28.8% for Control) but did decrease pregnancy loss (7.0% for CIDR and 15.6% for Control). The CIDR insert increased pregnancy per AI in cows with high progesterone at the time the CIDR insert was applied. Administration of a CIDR insert between Days 14 and 23 of the estrous cycle after first service did not increase overall pregnancy per AI to second service (24.7% and 22.7% for Resynch CIDR; 28.6% and 25.3% for Resynch control). For second service, RIDE cows had lower pregnancy rates in the Resynch CIDR group than in the Resynch control group. Cows with a CL (corpus luteum) at Day 30 had higher pregnancy rates in the Resynch CIDR group than those in the Resynch control group.     

Identification of Francisella tularensis Live Vaccine Strain CuZn Superoxide Dismutase as Critical for Resistance to Extracellularly Generated Reactive Oxygen Species

Francisella tularensis is an intracellular pathogen whose survival is in part dependent on its ability to resist the microbicidal activity of host-generated reactive oxygen species (ROS) and reactive nitrogen species (RNS). In numerous bacterial pathogens, CuZn-containing superoxide dismutases (SodC) are important virulence factors, localizing to the periplasm to offer protection from host-derived superoxide radicals (O₂⁻). In the present study, mutants of F. tularensis live vaccine strain (LVS) deficient in superoxide dismutases (SODs) were used to examine their role in defense against ROS/RNS-mediated microbicidal activity of infected macrophages. An in-frame deletion F. tularensis mutant of sodC (ΔsodC) and a F. tularensis ΔsodC mutant with attenuated Fe-superoxide dismutase (sodB) gene expression (sodB ΔsodC) were constructed and evaluated for susceptibility to ROS and RNS in gamma interferon (IFN-γ)-activated macrophages and a mouse model of respiratory tularemia. The F. tularensis ΔsodC and sodB ΔsodC mutants showed attenuated intramacrophage survival in IFN-γ-activated macrophages compared to the wild-type F. tularensis LVS. Transcomplementing the sodC gene in the ΔsodC mutant or inhibiting the IFN-γ-dependent production of O₂⁻ or nitric oxide (NO) enhanced intramacrophage survival of the sod mutants. The ΔsodC and sodB ΔsodC mutants were also significantly attenuated for virulence in intranasally challenged C57BL/6 mice compared to the wild-type F. tularensis LVS. As observed for macrophages, the virulence of the ΔsodC mutant was restored in ifn-γ^−/−, inos^−/−, and phox^−/− mice, indicating that SodC is required for resisting host-generated ROS. To conclude, this study demonstrates that SodB and SodC act to confer protection against host-derived oxidants and contribute to intramacrophage survival and virulence of F. tularensis in mice.Francisella tularensis is considered a potential biological threat due to its extreme infectivity, ease of artificial dissemination via aerosols, and substantial capacity to cause illness and death. A hallmark of all F. tularensis subspecies is their ability to survive and replicate within macrophages (18) and other cell types (6, 11, 25, 28). While recent work has furthered our understanding of F. tularensis virulence mechanisms, little is known with respect to its ability to resist the microbicidal production of reactive oxygen species (ROS) or reactive nitrogen species (RNS).Superoxide dismutases (SODs) are metalloproteins that are classified according to their coordinating active site metals. SODs catalyze the dismutation of the highly reactive superoxide (O₂⁻) anion to hydrogen peroxide (H₂O₂) and O₂ (26). The dismutation of O₂⁻ prevents accumulation of microbicidal ROS and RNS in infected macrophages. Three major categories of SODs have been identified in bacteria and include Mn-, Fe-, and CuZn-containing SODs (SodA, SodB, and SodC, respectively) and are required for aerobic survival (27). The F. tularensis genome encodes SodB (FTL_1791) and SodC (FTL_0380). In several intracellular bacterial pathogens, SodC is an important virulence factor, and its localization to the periplasmic space protects bacteria from host-derived O₂⁻ and NO radicals (8, 9, 21, 32). Moreover, many virulent bacteria possess two copies of the sodC gene (4). The evolutionary maintenance of an extra sodC gene copy suggests that it serves some essential function in survival (4). As an intracellular pathogen, F. tularensis is exposed to ROS and RNS generated by inflammatory cells during the macrophage activation process, which suggests that SODs may play an important role in its intracellular survival and pathogenesis. We have demonstrated that decreases in SodB activity render F. tularensis sensitive to ROS and attenuate virulence in mice (2). However, the contribution of F. tularensis SodC in virulence and intramacrophage survival has not been defined. In this study we have constructed a F. tularensis sodC mutant (ΔsodC) and a F. tularensis sodBC double mutant (sodB ΔsodC) and determined that SodC in conjunction with SodB primarily protects the pathogen from host-derived ROS and is required for intramacrophage survival and virulence of F. tularensis in mice.     

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.     

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.