bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of conformational free energies of peptides by multidimensional adaptive umbrella sampling
Authors: Wang Jun , Gu Yan , Liu Haiyan
Published: September 2006

We improve the multidimensional adaptive umbrella sampling method for the computation of conformational free energies of biomolecules. The conformational transition between the α-helical and β-hairpin conformational states of an alanine decapeptide is used as an example. Convergence properties of the weighted-histogram-analysis-based adaptive umbrella sampling can be improved by using multiple replicas in each adaptive iteration and by using adaptive updating of the bounds of the umbrella potential. Using positional root-mean-square deviations from structures of the α-helical and β-hairpin reference states as reaction coordinates, we obtained well-converged free energy surfaces of both the in-vacuum and in-solution decapeptide systems. From the free energy surfaces well-converged relative free energies between the two conformational states can be derived. Advantages and disadvantages of different methods for obtaining conformational free energies as well as implications of our results in studying conformational transitions of proteins and in improving force field are discussed.
Homogeneous cooperative upconversion in sol-gel fabricated erbium doped channel waveguides
Authors: Thomson R. R. , Bookey H. T. , Suyal N. , Kar A. K.
Published: September 2006

Time resolved fluorescence emission from erbium doped silicate channel waveguides is studied using an established model for homogeneous cooperative upconversion. Each sample was fabricated using only a single sol-gel deposition for each of the core and cladding layers. As a result of the analysis, values of 1.7×10−17 and 1.4×10−17cm3∕s are reported for the homogeneous cooperative upconversion constant that governs the interaction of homogeneously distributed erbium ions in the I13∕24 state. The values reported here are found to be in agreement with those reported in other predominantly silica based glasses.
High Q defect mode and laser action in one-dimensional hybrid photonic crystal containing cholesteric liquid crystal
Authors: Matsuhisa Yuko , Ozaki Ryotaro , Yoshino Katsumi , Ozaki Masanori
Published: September 2006

The authors have investigated the optical characteristics of a one-dimensional hybrid photonic crystal (1D HPC) containing cholesteric liquid crystal (CLC) as a defect by theoretical calculation and predicted the appearance of additional modes at the band edges of the CLC defect, whose Q factor was higher than those of the other defect modes. They have confirmed the appearance of the additional mode experimentally. Single-mode laser action with low pumping threshold was observed in a 1D HPC with a dye-doped CLC defect, which is based on the additional defect mode with a high Q factor peculiar to the CLC defect having periodic structure.
Efficient evaluation of short-range Hartree-Fock exchange in large molecules and periodic systems
Authors: Izmaylov Artur F. , Scuseria Gustavo E. , Frisch Michael J.
Published: September 2006

We present an efficient algorithm for the evaluation of short-range Hartree-Fock exchange energies and geometry gradients in Gaussian basis sets. Our method uses a hierarchy of screening levels to eliminate negligible two-electron integrals whose evaluation is the fundamental computational bottleneck of the procedure. By applying our screening technique to the Heyd-Scuseria-Ernzerhof [J. Chem. Phys. 118, 8207 (2003)] short-range Coulomb hybrid density functional, we achieve a computational efficiency comparable with that of standard nonhybrid density functional calculations.
Effect of Collagen-I Modified Composites on Proliferation and Differentiation of Human Alveolar Osteoblasts
Authors: Zhou Yefang , Hutmacher Dietmar W. , Varawan Sae-Lim , Lim Tit Meng
Published: September 2006

Collagen modification of scaffolds has been reported to promote matrix mineralization as an effective way to increase osseointegration of implants. The aim of this study was to investigate in vitro proliferation and differentiation of human alveolar osteoblasts (AOs) on medical-grade polycaprolactone–tricalcium phosphate (mPCL-TCP 80:20) scaffolds after collagen modification (mPCL-TCP-c) for 28 days. Collagen modification significantly increased the scaffold’s protein adsorption ability, and improved the initial seeding efficiency and cell attachment at day 1, compared with non-collagen-modified scaffolds. However, the total DNA content of both groups reached similar levels with no significant difference at 28 days’ culture. AOs were observed to spread along the collagen fibres and form extensive collagenous fibres with mineral nodules embedded, while multilayered cell sheets were formed in mPCL-TCP scaffolds. During culture, alkaline phosphatase (ALP) activity increased three- to five-fold in both groups, and collagen modification did not significantly affect either the metabolic rate or ALP activity kinetics of AOs. During osteogenic differentiation, similar gene expression of collagen type-I, osterix, osteopontin, and osteocalcin were detected in both groups. The mPCL-TCP group showed better organized mineralized tissue, but the mPCL-TCP-c showed more scattered and unorganized tissue. These results indicate that collagen modification improved the scaffold’s protein adsorption ability and encouraged initial cell attachment and distribution, but promoted fibrous-like tissue formation rather than mineralized tissue.
Gold-Loaded Organic/Inorganic Nanocomposite Honeycomb Membranes
Authors: Wong Kok Hou , Davis Thomas P. , Barner-Kowollik Christopher , Stenzel Martina H.
Published: September 2006

RAFT polymerization was used to prepare polystyrene – poly(4-vinyl pyridine) block copolymers, PSn-b-P(4VP)m. Well-defined block copolymers were obtained despite some indications of hydrolysis of the RAFT endgroup during synthesis. The block copolymer PS70-b-P(4VP)55 was self-assembled into micellar structures in dichloromethane, leading to nanoparticles with hydrodynamic diameters of 70 nm. The micelles were loaded with HAuCl4 and, upon reduction, micellar gold-containing nanoparticles with hydrodynamic diameters of 240 nm were obtained. These nanoparticles were employed in the preparation of honeycomb-structured porous films by means of the breath figures technique to yield gold nanocomposites with a hexagonal porous array.
Intersubunit signaling in glutamate-1-semialdehyde-aminomutase
Authors: Stetefeld J. , Jenny M. , Burkhard P.
Published: September 2006

Enzymes are highly dynamic and tightly controlled systems. However, allosteric communication linked to catalytic turnover is poorly understood. We have performed an integrated approach to trap several catalytic intermediates in the α2-dimeric key enzyme of chlorophyll biosynthesis, glutamate-1-semialdehyde aminomutase. Our data reveal an active-site “gating loop,” which undergoes a dramatic conformational change during catalysis, that is simultaneously open in one subunit and closed in the other. This loop movement requires a β-sheet-to-α-helix transition to assume the closed conformation, thus facilitating transport of substrate toward, and concomitantly forming, an integral part of the active site. The accompanying intersubunit cross-talk, which controls negative cooperativity between the allosteric pair, was explored at the atomic level. The central elements of the communication triad are the cofactor bound to different catalytic intermediates, the interface helix, and the gating loop. Together, they form a molecular switch in which the cofactor acts as a central signal transmitter linking the subunit interface with the gating loop.
Loss of Apc allows phenotypic manifestation of the transforming properties of an endogenous K-
ras
oncogene
in vivo
Authors: Sansom Owen J. , Meniel Valerie , Wilkins Julie A. , Cole Alicia M. , Oien Karin A. , Marsh Victoria , Jamieson Thomas J. , Guerra Carmen , Ashton Gabrielle H. , Barbacid Mariano , Clarke Alan R.
Published: September 2006

Oncogenic mutations in the K-
ras
gene occur in ≈50% of human colorectal cancers. However, the precise role that K-
ras
oncogenes play in tumor formation is still unclear. To address this issue, we have conditionally expressed an oncogenic K-
ras
V12
allele in the small intestine of adult mice either alone or in the context of Apc deficiency. We found that expression of K-
ras
V12
does not affect normal intestinal homeostasis or the immediate phenotypes associated with Apc deficiency. Mechanistically we failed to find activation of the Raf/MEK/ERK pathway, which may be a consequence of the up-regulation of a number of negative feedback loops. However, K-
ras
V12
expression accelerates intestinal tumorigenesis and confers invasive properties after Apc loss over the long term. In renal epithelium, expression of the oncogenic K-
ras
V12
allele in the absence of Apc induces the rapid development of renal carcinoma. These tumors, unlike those of intestinal origin, display activation of the Raf/MEK/ERK and Akt signaling pathways. Taken together, these data indicate that normal intestinal and kidney epithelium are resistant to malignant transformation by an endogenous K-
ras
oncogene. However, activation of K-
ras
V12
after Apc loss results in increased tumorigenesis with distinct kinetics. Whereas the effect of K-
ras
oncogenes in the intestine can been observed only after long latencies, they result in rapid carcinogenesis in the kidney epithelium. These data imply a window of opportunity for anti-K-ras therapies after tumor initiation in preventing tumor growth and invasion.
Cyclobutane pyrimidine dimers are predominant DNA lesions in whole human skin exposed to UVA radiation
Authors: Mouret Stéphane , Baudouin Caroline , Charveron Marie , Favier Alain , Cadet Jean , Douki Thierry
Published: September 2006

Solar UV radiation is the most important environmental factor involved in the pathogenesis of skin cancers. The well known genotoxic properties of UVB radiation (290–320 nm) mostly involve bipyrimidine DNA photoproducts. In contrast, the contribution of more-abundant UVA radiation (320–400 nm) that are not directly absorbed by DNA remains poorly understood in skin. Using a highly accurate and quantitative assay based on HPLC coupled with tandem mass spectrometry, we determined the type and the yield of formation of DNA damage in whole human skin exposed to UVB or UVA. Cyclobutane pyrimidine dimers, a typical UVB-induced DNA damage, were found to be produced in significant yield also in whole human skin exposed to UVA through a mechanism different from that triggered by UVB. Moreover, the latter class of photoproducts is produced in a larger amount than 8-oxo-7,8-dihydro-2′-deoxyguanosine, the most common oxidatively generated lesion, in human skin. Strikingly, the rate of removal of UVA-generated cyclobutane pyrimidine dimers was lower than those produced by UVB irradiation of skin. Finally, we compared the formation yields of DNA damage in whole skin with those determined in primary cultures of keratinocytes isolated from the same donors. We thus showed that human skin efficiently protects against UVB-induced DNA lesions, whereas very weak protection is afforded against UVA. These observations emphasize the likely role played by the UVA-induced DNA damage in skin carcinogenesis and should have consequences for photoprotection strategies.
Effective cell and gene therapy in a murine model of Gaucher disease
Authors: Enquist Ida Berglin , Nilsson Eva , Ooka Andreas , Månsson Jan-Eric , Olsson Karin , Ehinger Mats , Brady Roscoe O. , Richter Johan , Karlsson Stefan
Published: September 2006

Gaucher disease (GD) is a lysosomal storage disorder due to an inherited deficiency in the enzyme glucosylceramidase (GCase) that causes hepatosplenomegaly, cytopenias, and bone disease as key clinical symptoms. Previous mouse models with GCase deficiency have been lethal in the perinatal period or viable without displaying the clinical features of GD. We have generated viable mice with characteristic clinical symptoms of type 1 GD by conditionally deleting GCase exons 9–11 upon postnatal induction. Both transplantation of WT bone marrow (BM) and gene therapy through retroviral transduction of BM from GD mice prevented development of disease and corrected an already established GD phenotype. The gene therapy approach generated considerably higher GCase activity than transplantation of WT BM. Strikingly, both therapeutic modalities normalized glucosylceramide levels and practically no infiltration of Gaucher cells could be observed in BM, spleen, and liver, demonstrating correction at 5–6 months after treatment. The findings demonstrate the feasibility of gene therapy for type 1 GD
in vivo
. Our type 1 GD mice will serve as an excellent tool in the continued efforts toward development of safe and efficient cell and gene therapy for type 1 GD.
Estimating the diversity of dinosaurs
Authors: Wang Steve C. , Dodson Peter
Published: September 2006

Despite current interest in estimating the diversity of fossil and extant groups, little effort has been devoted to estimating the diversity of dinosaurs. Here we estimate the diversity of nonavian dinosaurs at ≈1,850 genera, including those that remain to be discovered. With 527 genera currently described, at least 71% of dinosaur genera thus remain unknown. Although known diversity declined in the last stage of the Cretaceous, estimated diversity was steady, suggesting that dinosaurs as a whole were not in decline in the 10 million years before their ultimate extinction. We also show that known diversity is biased by the availability of fossiliferous rock outcrop. Finally, by using a logistic model, we predict that 75% of discoverable genera will be known within 60–100 years and 90% within 100–140 years. Because of nonrandom factors affecting the process of fossil discovery (which preclude the possibility of computing realistic confidence bounds), our estimate of diversity is likely to be a lower bound.
Microbial community in a sediment-hosted CO
2
lake of the southern Okinawa Trough hydrothermal system
Authors: Inagaki Fumio , Kuypers Marcel M. M. , Tsunogai Urumu , Ishibashi Jun-ichiro , Nakamura Ko-ichi , Treude Tina , Ohkubo Satoru , Nakaseama Miwako , Gena Kaul , Chiba Hitoshi , Hirayama Hisako , Nunoura Takuro , Takai Ken , Jørgensen Bo B. , Horikoshi Koki , Boetius Antje
Published: September 2006

Increasing levels of CO
2
in the atmosphere are expected to cause climatic change with negative effects on the earth's ecosystems and human society. Consequently, a variety of CO
2
disposal options are discussed, including injection into the deep ocean. Because the dissolution of CO
2
in seawater will decrease ambient pH considerably, negative consequences for deep-water ecosystems have been predicted. Hence, ecosystems associated with natural CO
2
reservoirs in the deep sea, and the dynamics of gaseous, liquid, and solid CO
2
in such environments, are of great interest to science and society. We report here a biogeochemical and microbiological characterization of a microbial community inhabiting deep-sea sediments overlying a natural CO
2
lake at the Yonaguni Knoll IV hydrothermal field, southern Okinawa Trough. We found high abundances (>10
9
cm
−3
) of microbial cells in sediment pavements above the CO
2
lake, decreasing to strikingly low cell numbers (10
7
cm
−3
) at the liquid CO
2
/CO
2
-hydrate interface. The key groups in these sediments were as follows: (
i
) the anaerobic methanotrophic archaea ANME-2c and the Eel-2 group of Deltaproteobacteria and (
ii
) sulfur-metabolizing chemolithotrophs within the Gamma- and Epsilonproteobacteria. The detection of functional genes related to one-carbon assimilation and the presence of highly
13
C-depleted archaeal and bacterial lipid biomarkers suggest that microorganisms assimilating CO
2
and/or CH
4
dominate the liquid CO
2
and CO
2
-hydrate-bearing sediments. Clearly, the Yonaguni Knoll is an exceptional natural laboratory for the study of consequences of CO
2
disposal as well as of natural CO
2
reservoirs as potential microbial habitats on early Earth and other celestial bodies.
Anterior cingulate cortex activity can be independent of response conflict in Stroop-like tasks
Authors: Roelofs Ardi , van Turennout Miranda , Coles Michael G. H.
Published: September 2006

Cognitive control includes the ability to formulate goals and plans of action and to follow these while facing distraction. Previous neuroimaging studies have shown that the presence of conflicting response alternatives in Stroop-like tasks increases activity in dorsal anterior cingulate cortex (ACC), suggesting that the ACC is involved in cognitive control. However, the exact nature of ACC function is still under debate. The prevailing conflict detection hypothesis maintains that the ACC is involved in performance monitoring. According to this view, ACC activity reflects the detection of response conflict and acts as a signal that engages regulative processes subserved by lateral prefrontal brain regions. Here, we provide evidence from functional MRI that challenges this view and favors an alternative view, according to which the ACC has a role in regulation itself. Using an arrow–word Stroop task, subjects responded to incongruent, congruent, and neutral stimuli. A critical prediction made by the conflict detection hypothesis is that ACC activity should be increased only when conflicting response alternatives are present. Our data show that ACC responses are larger for neutral than for congruent stimuli, in the absence of response conflict. This result demonstrates the engagement of the ACC in regulation itself. A computational model of Stroop-like performance instantiating a version of the regulative hypothesis is shown to account for our findings.
Engineering RNA sequence specificity of Pumilio repeats
Authors: Cheong Cheom-Gil , Hall Traci M. Tanaka
Published: September 2006

Puf proteins bind RNA sequence specifically and regulate translation and stability of target mRNAs. A “code” for RNA recognition has been deduced from crystal structures of the Puf protein, human Pumilio1, where each of eight repeats binds an RNA base via a combination of three side chains at conserved positions. Here, we report the creation of seven soluble mutant proteins with predictably altered sequence specificity, including one that binds tightly to adenosine-uracil-rich element RNA. These data show that Pumilio1 can be used as a scaffold to engineer RNA-binding proteins with designed sequence specificity.
Lifelong learning and the knowledge economy: those that know and those that do not—the discourse of the European Union
Authors: Brine Jacky
Published: September 2006

This article is based on a textual analysis of European Commission documents that, from 1993 to 2006, construct the discourses of lifelong learning and the knowledge economy. Exploring an apparent conceptual laxity, it finds absolute consistency in the construction of two categories of learner: the high knowledge‐skilled learner (graduate/postgraduate) for the knowledge economy, and the low knowledge‐skilled learner located in (or beyond) the knowledge society. The low knowledge‐skilled learners are not only those at risk, they are increasingly constructed as the risk. The analysis suggests that the binary classification is initially classed and raced—and only then is it gendered. In contrast, labour market studies of the knowledge economy, providing either gendered or national data, obscure the vital cross‐cutting matrix of social class, ‘race’ and age. The article advocates further studies of lifelong learning practices and labour market data based on finely‐crossed analyses of social class, poverty, age and race.
Lifting the hood: lifelong learning and young, white, provincial working‐class masculinities
Authors: Quinn Jocey , Thomas Liz , Slack Kim , Casey Lorraine , Thexton Wayne , Noble John
Published: September 2006

Young, white, provincial working‐class men are portrayed as a threat to lifelong learning goals. They are least likely to enter university and most likely to ‘drop out’. However, white provincial masculinities are neglected in debates on gender and lifelong learning. This article uses a UK‐wide study of working‐class ‘drop‐out’ to explore the situated nature of such masculinities, how they are performed by students and consumed by others and reproduced by university cultures and pedagogies. It concludes that such students struggle to fit the fluid paradigm of the new lifelong learner and are constantly being fixed in place by structural inequality, discursive frames and institutional practices. Their ‘drop‐out’ is shaped by masculinity, but need not be viewed pejoratively. It can be a frustrated search for lifelong learning, often inspired by a love of informal learning. This should be respected, not ignored.
PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection
Authors: Petrovas Constantinos , Casazza Joseph P. , Brenchley Jason M. , Price David A. , Gostick Emma , Adams William C. , Precopio Melissa L. , Schacker Timothy , Roederer Mario , Douek Daniel C. , Koup Richard A.
Published: September 2006

Here, we report on the expression of programmed death (PD)-1 on human virus-specific CD8+ T cells and the effect of manipulating signaling through PD-1 on the survival, proliferation, and cytokine function of these cells. PD-1 expression was found to be low on naive CD8+ T cells and increased on memory CD8+ T cells according to antigen specificity. Memory CD8+ T cells specific for poorly controlled chronic persistent virus (HIV) more frequently expressed PD-1 than memory CD8+ T cells specific for well-controlled persistent virus (cytomegalovirus) or acute (vaccinia) viruses. PD-1 expression was independent of maturational markers on memory CD8+ T cells and was not directly associated with an inability to produce cytokines. Importantly, the level of PD-1 surface expression was the primary determinant of apoptosis sensitivity of virus-specific CD8+ T cells. Manipulation of PD-1 led to changes in the ability of the cells to survive and expand, which, over several days, affected the number of cells expressing cytokines. Therefore, PD-1 is a major regulator of apoptosis that can impact the frequency of antiviral T cells in chronic infections such as HIV, and could be manipulated to improve HIV-specific CD8+ T cell numbers, but possibly not all functions in vivo.
Non-persistently recurrent points, qc-surgery and instability of rational maps with totally disconnected Julia sets
Authors: Makienko Peter
Published: September 2006

Let

R
R

be a rational map with a totally disconnected Julia set

J
(
R
)

J(R)

. If the postcritical set on

J
(
R
)

J(R)

contains a non-persistently recurrent (or conical) point, then we show that the map

R
R

cannot be a structurally stable map.
Targeted projection pursuit for visualizing gene expression data classifications
Authors: Faith Joe , Mintram Robert , Angelova Maia
Published: September 2006

We present a novel method for finding low-dimensional views of high-dimensional data: Targeted Projection Pursuit. The method proceeds by finding projections of the data that best approximate a target view. Two versions of the method are introduced; one version based on Procrustes analysis and one based on an artificial neural network. These versions are capable of finding orthogonal or non-orthogonal projections, respectively. The method is quantitatively and qualitatively compared with other dimension reduction techniques. It is shown to find 2D views that display the classification of cancers from gene expression data with a visual separation equal to, or better than, existing dimension reduction techniques.

Availability: source code, additional diagrams, and original data are available from

Contact:  joe.faith@unn.ac.uk

Supplementary information: Supplementary data are available at Bioinformatics online.
Discovering disease-genes by topological features in human protein–protein interaction network
Authors: Xu Jianzhen , Li Yongjin
Published: September 2006

Motivation: Mining the hereditary disease-genes from human genome is one of the most important tasks in bioinformatics research. A variety of sequence features and functional similarities between known human hereditary disease-genes and those not known to be involved in disease have been systematically examined and efficient classifiers have been constructed based on the identified common patterns. The availability of human genome-wide protein–protein interactions (PPIs) provides us with new opportunity for discovering hereditary disease-genes by topological features in PPIs network.

Results: This analysis reveals that the hereditary disease-genes ascertained from OMIM in the literature-curated (LC) PPIs network are characterized by a larger degree, tendency to interact with other disease-genes, more common neighbors and quick communication to each other whereas those properties could not be detected from the network identified from high-throughput yeast two-hybrid mapping approach (EXP) and predicted interactions (PDT) PPIs network. KNN classifier based on those features was created and on average gained overall prediction accuracy of 0.76 in cross-validation test. Then the classifier was applied to 5262 genes on human genome and predicted 178 novel disease-genes. Some of the predictions have been validated by biological experiments.

Contact:  jianzxu@hotmail.com

Supplementary information: Supplementary data are available at Bioinformatics online.
Description of Sphingobium fuliginis sp. nov., a phenanthrene-degrading bacterium from a fly ash dumping site, and reclassification of Sphingomonas cloacae as Sphingobium cloacae comb. nov.
Authors: Prakash Om , Lal Rup
Published: September 2006

A phenanthrene-degrading bacterium, strain TKPT, was isolated from a fly ash dumping site of the thermal power plant in Panki, Kanpur, India, by an enrichment culture method using phenanthrene as the sole source of carbon and energy. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain belonged to the genusSphingobium, as it showed highest sequence similarity toSphingobium herbicidovoransDSM 11019T(97.3 %) andSphingomonas cloacaeJCM 10874T(96.5 %), compared with only 91–93 % similarity to members of other genera such asSphingomonas sensu stricto,Novosphingobium,SphingopyxisandSphingosinicella. In DNA–DNA hybridization experiments with strains that were closely related phylogenetically and in terms of 16S rRNA gene sequences, i.e.Sphingobium herbicidovoransDSM 11019TandSphingomonas cloacaeJCM 10874T, strain TKPTshowed less than 70 % relatedness. Strain TKPTcontained sphingoglycolipids SGL-1 and SGL-2 and 18 : 1ω7cas the predominant fatty acid, with 16 : 0 as a minor component and 14 : 0 2-OH as the major 2-hydroxy fatty acid. Thus, phylogenetic analysis, DNA–DNA hybridization, fatty acid and polar lipid profiles and differences in physiological and morphological features from the most closely related members of theSphingobiumgroup showed that strain TKPTrepresents a distinct species ofSphingobium. The nameSphingobium fuliginissp. nov. is proposed, with the type strain TKPT(=MTCC 7295T=CCM 7327T).Sphingomonas cloacaeJCM 10874Tformed a coherent cluster with members ofSphingobium, did not reduce nitrate to nitrite and had a fatty acid profile similar to those ofSphingobiumspecies; henceSphingomonas cloacaeshould be transferred to the genusSphingobiumasSphingobium cloacaecomb. nov., with the type strain JCM 10874T(=DSM 14926T).
Sphingomonas dokdonensis sp. nov., isolated from soil
Authors: Yoon Jung-Hoon , Lee Mi-Hwa , Kang So-Jung , Lee Soo-Young , Oh Tae-Kwang
Published: September 2006

A Gram-negative, rod-shaped,Sphingomonas-like bacterial strain, DS-4T, was isolated from soil of Dokdo, Korea, and its taxonomic position was investigated using a polyphasic approach. Strain DS-4Tgrew optimally on trypticase soy agar medium without NaCl at pH 6.0–6.5 and 25 °C. It contained Q-10 as the predominant ubiquinone and C18 : 1ω7c, C16 : 0, C14 : 02-OH and C16 : 1ω7cand/or iso-C15 : 02-OH as the major fatty acids. Sphingoglycolipid, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and unidentified phospholipid were the major polar lipids. The DNA G+C content was 66.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DS-4Tfell within the evolutionary radiation comprisingSphingomonasspecies. Levels of 16S rRNA gene sequence similarity between strain DS-4Tand the type strains ofSphingomonasspecies ranged from 93.0 to 97.6 %. DNA–DNA relatedness data and differential phenotypic properties, together with the phylogenetic distinctiveness, demonstrated that strain DS-4Tdiffers from the recognizedSphingomonasspecies. On the basis of phenotypic, phylogenetic and genetic data, this strain represents a novel species of the genusSphingomonas, for which the nameSphingomonas dokdonensissp. nov. is proposed, with DS-4T(=KCTC 12541T=CIP 108841T) as the type strain.
Rheinheimera perlucida sp. nov., a marine bacterium of the Gammaproteobacteria isolated from surface water of the central Baltic Sea
Authors: Brettar Ingrid , Christen Richard , Höfle Manfred G.
Published: September 2006

A bacterial isolate from the Baltic Sea, BA131T, was characterized for its physiological and biochemical features, fatty acid profile, G+C content and phylogenetic position based on comparative 16S rRNA gene sequence analysis. The strain was isolated from surface water of the central Baltic Sea during the decay of a plankton bloom. Phylogenetic analyses of the 16S rRNA gene sequence revealed a clear affiliation with theGammaproteobacteria, and showed closest phylogenetic relationships with the generaAlishewanellaandRheinheimera. The G+C content of the DNA of strain BA131Twas 48.9 mol%. Cells were non-pigmented, Gram-negative, rod-shaped, motile by means of a single polar flagellum and catalase- and oxidase-positive. Growth was observed at salinities from 0 to 8 %, with an optimum at 1–3 %. Temperature for growth ranged from 4 to 37 °C, with an optimum around 25 °C. The fatty acids were dominated by 16 : 0 (17–18 %) and by unsaturated compounds (>61 % of the total): 16 : 1ω7c(24–33 %), 17 : 1ω8c(14–18 %) and 18 : 1ω7c(9–12 %). Based on the data presented, BA131Tis proposed as the type strain of a novel species of the genusRheinheimera,Rheinheimera perlucidasp. nov. The type strain is BA131T(=LMG 23581T=CIP 109200T).
Mycobacterium colombiense sp. nov., a novel member of the Mycobacterium avium complex and description of MAC-X as a new ITS genetic variant
Authors: Murcia Martha I. , Tortoli Enrico , Menendez M. Carmen , Palenque Elia , Garcia Maria J.
Published: September 2006

Forty-five mycobacterial strains isolated from 23 Colombian HIV-positive patients were identified as members of theMycobacterium aviumcomplex (MAC) and were characterized using different molecular approaches. Seven of the isolates showed characteristic features that allowed them to be differentiated from other members of the complex. The isolates had a novel 16S–23S rRNA internal transcribed spacer (ITS 1) gene sequence which is described as a new sequevar, MAC-X. All of the seven novel isolates gave a positive result with the MAC-specific AccuProbe (Gen-Probe), but tested negative forMycobacterium aviumandMycobacterium intracellularespecies-specific probes (64 and 100 % of the isolates, respectively). The novel isolates could be differentiated phenotypically from other members of the MAC on the basis of the production of urease and by a consistent mycolic acid pattern. The novel isolates shared some characteristics withM. avium, such as the avium variant I (av-I) pattern of thehsp65 gene as determined by PCR restriction analysis and a positive PCR result for themig(macrophage-induced) gene. However, the novel isolates showed a unique 16S rRNA gene sequence. DNA–DNA relatedness values, from 24 to 44 %, confirmed the distinction of the novel isolates from other members of the MAC at the genetic level and their status as members of a separate species. The novel isolates are proposed as representatives of a novel species,Mycobacterium colombiensesp. nov., that is closely related toM. aviumwithin the MAC. The type strain is 10BT(=CIP 108962T=CECT 3035T).
Reclassification of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis as Aggregatibacter actinomycetemcomitans gen. nov., comb. nov., Aggregatibacter aphrophilus comb. nov. and Aggregatibacter segnis comb. nov., and emended description of Aggregatibacter aphrophilus to include V factor-dependent and V factor-independent isolates
Authors: Nørskov-Lauritsen Niels , Kilian Mogens
Published: September 2006

The aim of this study was to reinvestigate the relationships and the generic affiliations of the species Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis. The nicotinamide phosphoribosyltransferase gene (nadV) conferring V factor-independent growth was identified in Haemophilus aphrophilus. The gene encodes a polypeptide of 462 amino acids that shows 74.5 % amino acid sequence identity to the corresponding enzyme from Actinobacillus actinomycetemcomitans. Ten isolates of Haemophilus paraphrophilus all carried a nadV pseudogene. DNA from Haemophilus aphrophilus was able to transform Haemophilus paraphrophilus into the NAD-independent phenotype. The transformants carried a full-length nadV inserted in the former locus of the pseudogene. The DNA–DNA relatedness between the type strains of Haemophilus aphrophilus and Haemophilus paraphrophilus was 77 %. We conclude that the division into two species Haemophilus aphrophilus and Haemophilus paraphrophilus is not justified and that Haemophilus paraphrophilus should be considered a later heterotypic synonym of Haemophilus aphrophilus. Forty strains of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus segnis were investigated by multilocus sequence analysis. The 40 strains form a monophyletic group clearly separate from other evolutionary lineages of the family Pasteurellaceae. We propose the transfer of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus segnis to a new genus Aggregatibacter gen. nov. as Aggregatibacter actinomycetemcomitans comb. nov. (the type species; type strain ATCC 33384T=CCUG 13227T=CIP 52.106T=DSM 8324T=NCTC 9710T), Aggregatibacter aphrophilus comb. nov. (type strain ATCC 33389T=CCUG 3715T=CIP 70.73T=NCTC 5906T) and Aggregatibacter segnis comb. nov. (type strain HK316T=ATCC 33393T=CCUG 10787T=CCUG 12838T=CIP 103292T=NCTC 10977T). The species of the genus Aggregatibacter are independent of X factor and variably dependent on V factor for growth in vitro.
Metschnikowia sinensis sp. nov., Metschnikowia zizyphicola sp. nov. and Metschnikowia shanxiensis sp. nov., novel yeast species from jujube fruit
Authors: Xue Meng-Lin , Zhang Li-Qun , Wang Qi-Ming , Zhang Ji-Shu , Bai Feng-Yan
Published: September 2006

Eight yeast strains were isolated from jujube fruit surfaces collected in Shanxi and Shandong Provinces, China. All eight strains produced needle-shaped ascospores under suitable conditions. Three separate groups, representing three novel species in the genus Metschnikowia, were recognized by sequence comparisons of the 26S rDNA D1/D2 domain and internal transcribed spacer (ITS) region. The names Metschnikowia sinensis sp. nov. (type strain XY103T=AS 2.3110T=CBS 10357T), Metschnikowia zizyphicola sp. nov. (type strain XY201T=AS 2.3111T=CBS 10358T) and Metschnikowia shanxiensis sp. nov. (type strain XY801T=AS 2.3112T=CBS 10359T) are proposed for the three novel species. Phylogenetic analysis of the 26S rDNA D1/D2 domain sequence showed that these three novel species are clustered in a clade together with the previously described species Metschnikowia fructicola, Metschnikowia andauensis, Metschnikowia pulcherrima and Metschnikowia chrysoperlae.
Mutation of the Rev-binding loop in the human immunodeficiency virus 1 leader causes a replication defect characterized by altered RNA trafficking and packaging
Authors: Greatorex Jane S. , Palmer Elizabeth A. , Pomerantz Roger J. , Dangerfield John A. , Lever Andrew M. L.
Published: September 2006

An internal RNA loop, located within the packaging signal of human immunodeficiency virus 1, that resembles the Rev-responsive element (RRE) closely was identified previously. Subsequent in vitro studies confirmed that the loop, termed loop A, could bind Rev protein specifically. Its proximity to the major splice donor has suggested a role for Rev–loop A interaction supplementary to or preceding that of the Rev–RRE interaction. To investigate this further in a replication-competent provirus, loop A was mutated to decrease its affinity for Rev. Impairing the Rev–loop A interaction led to reduced nuclear export of viral genomic RNA. RNA packaging decreased by approximately 30 %. Viral protein production and export of virus particles appeared normal; however, the virus was severely replication-deficient. The loop A sequence, which is 98 % conserved amongst viral isolates, is implicated in several cis-acting functions critical to virus viability.
Titanium dioxide photocatalytic inactivation of prions
Authors: Paspaltsis Ioannis , Kotta Konstantia , Lagoudaki Roza , Grigoriadis Nikolaos , Poulios Ioannis , Sklaviadis Theodoros
Published: September 2006

Prions are postulated to be the infectious agents of a family of transmissible, fatal, neurodegenerative disorders affecting both humans and animals. The possibility of prion transmission constitutes a public-health risk that confronts regulatory authorities everywhere. The main problem in handling prions is the fact that they are extremely resistant to standard decontamination methods. Thus, the use of harsh and expensive practices to destroy prions is inevitable. The development of applicable and efficient prion-inactivation practices is still highly important for the prevention of accidental transmission. In the search for effective and environmentally friendly methods to eliminate organic compounds and bacteria, much attention has been focused on the so-called advanced oxidation processes. These are based on the formation of hydroxyl radicals, which are known to possess a high reductive potential. This study tested the potential of titanium dioxide, an inexpensive and completely inert reagent, to inactivate prions in a heterogeneous photocatalytic process. Initialin vitroexperiments were followed by a bioassay with the scrapie strain 263K in Syrian hamsters. The results obtained from this study indicate that titanium dioxide photocatalytic treatment of scrapie-infected brain homogenates reduces infectivity titres significantly.
Epstein–Barr virus nuclear antigen 3A contains six nuclear-localization signals
Authors: Buck Marion , Burgess Anita , Stirzaker Roslynn , Krauer Kenia , Sculley Tom
Published: September 2006

The Epstein–Barr nuclear antigen 3A (EBNA3A) is one of only six viral proteins essential for Epstein–Barr virus-induced transformation of primary human B cells in vitro. Viral proteins such as EBNA3A are able to interact with cellular proteins, manipulating various biochemical and signalling pathways to initiate and maintain the transformed state of infected cells. EBNA3A has been reported to have one nuclear-localization signal and is targeted to the nucleus during transformation, where it associates with components of the nuclear matrix. By using enhanced green fluorescent protein-tagged deletion mutants of EBNA3A in combination with site-directed mutagenesis, an additional five functional nuclear-localization signals have been identified in the EBNA3A protein. Two of these (aa 63–66 and 375–381) were computer-predicted, whilst the remaining three (aa 394–398, 573–578 and 598–603) were defined functionally in this study.
Human cellular protein VRK2 interacts specifically with Epstein–Barr virus BHRF1, a homologue of Bcl-2, and enhances cell survival
Authors: Li Long-Yuan , Liu Mei-Ying , Shih Hsiu-Ming , Tsai Ching-Hwa , Chen Jen-Yang
Published: September 2006

BHRF1, an early gene product of Epstein–Barr virus (EBV), is structurally and functionally homologous to Bcl-2, a cellular anti-apoptotic protein. BHRF1 has been shown to protect cells from apoptosis induced by numerous external stimuli. Nasopharyngeal carcinoma is an epithelial cancer associated closely with EBV infection. Specific proteins that might interact with and modulate the BHRF1 anti-apoptotic activity in normal epithelial cells are of interest. Therefore, a cDNA library derived from normal human foreskin keratinocytes was screened by the yeast two-hybrid system and a cellular gene encoding human vaccinia virus B1R kinase-related kinase 2 (VRK2) was isolated. Interaction between the cellular VRK2 and viral BHRF1 proteins was further demonstrated by glutathioneS-transferase pull-down assays, confocal laser-scanning microscopy and co-immunoprecipitation. Analyses of VRK2-deletion mutants revealed that a 108 aa fragment at the C terminus was important for VRK2 to interact with BHRF1. For BHRF1, aa 1–18 and 89–142 were crucial in interacting with VRK2 and these two regions are counterparts of Bcl-2 homology domains 4 and 1. Overexpressed VRK2 alone showed a modest effect in anti-apoptosis and appeared to enhance cell survival in the presence of BHRF1. However, this enhancement was not observed when VRK2 was co-expressed with Bcl-2. The results indicate that human VRK2 interacts specifically with EBV BHRF1 and that the interaction is involved in protecting cells from apoptosis.
Rubella virus pseudotypes and a cell–cell fusion assay as tools for functional analysis of the rubella virus E2 and E1 envelope glycoproteins
Authors: Claus Claudia , Hofmann Jörg , Überla Klaus , Liebert U. G.
Published: September 2006

The rubivirus Rubella virus contains the two envelope glycoproteins E2 and E1 as a heterodimeric spike complex embedded in its lipid envelope. The functions of both proteins, especially of E2, in the process of viral entry are still not entirely understood. In order to dissect E2 and E1 entry functions from post-entry steps, pseudotypes of lentiviral vectors based on Simian immunodeficiency virus were used. C-terminally modified E2 and E1 variants successfully pseudotyped lentiviral vector particles. This is the first report to show that not only E1, but also E2, is able to mediate infectious viral entry. Furthermore, a cell–cell fusion assay was used to further clarify membrane-fusion activities of E2 and E1 as one of the early steps of infection. It was demonstrated that the capsid protein, when coexpressed in cis, enhances the degree of E2- and E1-mediated cell–cell fusion.
Temporal and differential gene expression of Singapore grouper iridovirus
Authors: Chen Li Ming , Wang Fan , Song Wenjun , Hew Choy Leong
Published: September 2006

Singapore grouper iridovirus (SGIV), an iridovirus in the genus Ranavirus, is a major pathogen that results in significant economic losses in grouper aquaculture. To investigate further its infective mechanisms, for the first time, a viral DNA microarray was generated for the SGIV genome to measure the expression of its predicted open reading frames simultaneously in vitro. By using the viral DNA microarray, the temporal gene expression of SGIV was characterized and the DNA microarray data were consistent with the results of real-time RT-PCR studies. Furthermore, different-stage viral genes (i.e. immediate-early, early and late genes) of SGIV were uncovered by combining drug treatments and DNA microarray studies. These results should offer important insights into the replication and pathogenesis of iridoviruses.
MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant
Authors: Lu Cheng , Kulkarni Karthik , Souret Frédéric F. , MuthuValliappan Ramesh , Tej Shivakundan Singh , Poethig R. Scott , Henderson Ian R. , Jacobsen Steven E. , Wang Wenzhong , Green Pamela J. , Meyers Blake C.
Published: September 2006

The Arabidopsis genome contains a highly complex and abundant population of small RNAs, and many of the endogenous siRNAs are dependent on RNA-Dependent RNA Polymerase 2 (RDR2) for their biogenesis. By analyzing an rdr2 loss-of-function mutant using two different parallel sequencing technologies, MPSS and 454, we characterized the complement of miRNAs expressed in Arabidopsis inflorescence to considerable depth. Nearly all known miRNAs were enriched in this mutant and we identified 13 new miRNAs, all of which were relatively low abundance and constitute new families. Trans-acting siRNAs (ta-siRNAs) were even more highly enriched. Computational and gel blot analyses suggested that the minimal number of miRNAs in Arabidopsis is ∼155. The size profile of small RNAs in rdr2 reflected enrichment of 21-nt miRNAs and other classes of siRNAs like ta-siRNAs, and a significant reduction in 24-nt heterochromatic siRNAs. Other classes of small RNAs were found to be RDR2-independent, particularly those derived from long inverted repeats and a subset of tandem repeats. The small RNA populations in other Arabidopsis small RNA biogenesis mutants were also examined; a dcl2/3/4 triple mutant showed a similar pattern to rdr2, whereas dcl1–7 and rdr6 showed reductions in miRNAs and ta-siRNAs consistent with their activities in the biogenesis of these types of small RNAs. Deep sequencing of mutants provides a genetic approach for the dissection and characterization of diverse small RNA populations and the identification of low abundance miRNAs.
Synchronization of Mammalian Cell Cultures in Mitosis Using Selective Detachment
Authors: Galgano Paul J. , Schildkraut Carl L.
Published: September 2006

INTRODUCTION

This protocol describes a method for synchronizing monolayer cells in mitosis using selective detachment from their substrate. During mitosis, cells become more spherical, causing them to become more loosely attached to their substrate. The “rounded up” cells are selectively detached by tapping the culture flask, resulting in a population in which as many as 90-98% of the cells are in mitosis. The drug nocodazole is used to increase the percentage of cells undergoing mitosis before detachment is performed. This procedure has been applied to mouse fibroblast and CHO (Chinese hamster ovary) cells. Since different cell types may attach differently, it will be necessary to determine the amount of force needed to remove loosely attached cells and the mitotic cells.
Staining of Tissue Slices for Analysis of Axonal Pathfinding in dsRNA-Treated Avian Embryos
Authors: Stoeckli Esther T.
Published: September 2006

INTRODUCTION

This protocol describes a method to stain nerve fibers in tissue slices of avian embryos using an antibody against the 160-kD subunit of neurofilaments. This allows the comparison of the branching pattern of motor and sensory neurons between control and experimental embryos. The tissue is cut in slices using a vibratome or tissue slicer. The protocol is suitable for older embryos after approximately stage 33 and regions that are not accessible by whole-mount analysis. Slices can also be used for tracing axon tracts with lipophilic dyes rather than immunohistochemical detection of all neurites with anti-neurofilament antibodies.
Immunoprecipitation: Lysing Yeast Cells Using Glass Beads
Authors: Harlow Ed , Lane David
Published: September 2006

INTRODUCTION

For yeast, the preferred method of lysis is mechanical shearing by vortexing the cells in the presence of glass beads as described in this protocol. This is a powerful lysis method but imparts extensive energy into the lysis procedure and results in considerable denaturation of the samples. For this reason, yeast immunoprecipitations are not as powerful a technique as similar studies in mammalian cells and should not be relied upon for determination of protein properties that require authentic protein structure. The lysate prepared in this procedure is ready for preclearing.