bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermal and Starvation Effects on the Minimum Film Thickness in Inlet Zone in Cold Rolling
Authors: Singh P. , Pandey R. K. , Nath Y.
Published: May 2008

The main objective of this research is to analyze the variation of minimum film thickness in the inlet zone of roll-strip interface by incorporating starvation and viscous shear heating effects at high rolling speeds (5–20m∕s), reduction ratios (0.05–0.20), and slip values (varying up to 20%). An additional objective of this paper is to develop empirical relations for predictions of minimum film thicknesses (both isothermal and thermal) and maximum film temperature rise in the inlet zone of the lubricated roll strip contact as functions of roll-speed, reduction ratio, material parameter, slip, and starvation parameter. An efficient numerical method based on Lobatto quadrature technique is adopted for rigorous analysis of the present problem. The results reveal that the existence of starvation seems to be beneficial in terms of reduction in maximum film temperature rise as well as reduction in quantity of oil required for lubrication provided thin continuous film exists at the contact.
Rolling Stream Trails: An Alternative Cavitation
Analysis
Authors: Pan Coda H. T. , Kim Tae Ho , Rencis Joseph J.
Published: May 2008

The rolling stream trail (RST) model introduces a new formulation of cavitation analysis of an eccentric journal bearing; it is presented as a preferred foundation for cavitation analysis of a journal bearing to replace Floberg’s streamer hypothesis that had been adopted by many investigators in recent past. Based on a careful reinterpretation of published experimental photographic data, noting the blunt-nosed shape of the interspersing space that separates adjacent wetting outlines at the rupture boundary, it stipulates a 3D flow structure for transition from the filled fluid film (FFF) to a cross-void fluid transportation process. The transition starts as a two-component composite rupture front and becomes an adhered film (AF) that is masked by rolled-on stream trails, which are drawn from the rupture front. The AF moves with the journal surface across the void to feed the FFF at the formation boundary. Upon averaged across a full period of the rupture front, Olsson’s equation for flow continuity relative to a moving cavitation boundary yields a moving speed of the rupture front that is proportional to the reciprocal of the width fraction of the wet pockets multiplied into the FFF pressure gradient. For all nonvanishing width fraction of the wet pockets, both rupture and formation boundaries move with finite speeds. RST is an initial value time-dependent problem that deals with both the FFF and AF that are joined at rupture and formation boundaries. The initial fluid content in the void span is bracketed between a dry void and a freshly cavitated wet void. As time progresses, transportation of AF across the void space and boundary motions form a coupled evolution process. Formulas for the boundary motions indicate that the formation boundary would become stationary simultaneously as the rupture boundary approaches the Swift–Stieber condition. For implementation of RST analysis, analytic functions originally derived by Sommerfeld in his classical paper are employed to construct FFF pressure profile and subsequently to calculate boundary speeds. Precision and robustness achieved in this approach assure viability of RST cavitation analysis. Results include temporal evolution profiles of the FFF pressure and the cavitation boundary trajectories. Basic concepts and mathematical formulation of RST are applicable to 2D problems. A scheme to include squeeze film motion is outlined.
Dynamic Analysis of Rotor System With Misaligned Retainer Bearings
Authors: Kärkkäinen Antti , Helfert Marlene , Aeschlimann Beat , Mikkola Aki
Published: May 2008

Active magnetic bearings present a technology that has many advantages compared to traditional bearing concepts. Active magnetic bearings, however, require retainer bearings in order to prevent damages in the event of a component, power, or a control system failure. In the drop-down, when the rotor drops from the magnetic field on the retainer bearings, the design of the retainer bearings has a significant influence on the dynamic behavior of the rotor. In this study, the dynamics of an active magnetic bearing supported rotor during the drop on retainer bearings is studied employing a simulation model. The retainer bearings are modeled using a detailed ball bearing model while the flexibility of the rotor is described using the finite element method with component mode synthesis. The model is verified by comparing measurements carried out using an existing test rig and simulation results. In this study, the verified simulation model is employed studying the effect of misalignment of retainer bearings during the rotor drop-down on the retainer bearings. It is concluded in this study that the misalignment of the retainer bearings is harmful and can lead to whirling motion of the rotor.
Stresses, Singularities, and a Complementary Energy Principle for
Large Strain Elasticity
Authors: Gao Yu-Chen , Jin Ming , Dui Guan-Suo
Published: May 2008

This article reviews three aspects of large strain elasticity. First, various conjugate stress tensors to strain tensors are reviewed. Many researchers have studied the theory of large strain elasticity. Many stress tensors including the Cauchy stress tensor, the first and the second Piola–Kirchhoff stress tensor, and the Jaumann stress tensor have been proposed to describe the stress state at a point. Recently, the first author of this article proposed a concept of “base forces” to reveal the essence of stress state. By the concept of base forces, the description of the stress state becomes clearer than other stress tensors. We attempt to take base forces as a basic point of view to deal with a review in which different descriptions of stress state are discussed and compared. The governing equations and boundary conditions expressed by the base forces are given. Second, this article reviews the solution of some singularity problems for large strain elasticity, i.e., problems of stress singularity at a crack or a notch tip, at the point of application of a concentrated force and at the vertex of contact in rubberlike materials. Methods of getting the singularity index of stress by using base forces are introduced and compared to earlier work. Complementary energy principles for large strain elasticity have eluded researchers for nearly 100years. A review of some important advances in this is also given, and a new complementary energy principle related to base forces is introduced.
Fluid Flow in an Annular Microchannel Subjected to Uniform Wall Injections
Authors: Layeghi Mohammad , Seyf Hamid Reza
Published: May 2008

Analytical analysis of fluid flow in an annular microchannel subjected to uniform wall injection at various Reynolds numbers is presented. The classical Navier–Stokes equations are used in the present study. Mathematically, using an appropriate change of variable, Navier–Stokes equations are transformed to a set of nonlinear ordinary differential equations. The governing equations are analytically solved using series solution method. Some analytical results are given for the prediction of velocity profiles and pressure distributions in annular microchannels. The agreement between the computational fluid dynamics and the analytical predictions is very good. However, the analytical results are valid in a limited range of radius ratios and needs more study.
Phosphorylation of Retinoblastoma Protein by Viral Protein with Cyclin-Dependent Kinase Function
Authors: Hume Adam J. , Finkel Jonathan S. , Kamil Jeremy P. , Coen Donald M. , Culbertson Michael R. , Kalejta Robert F.
Published: May 2008

As obligate intracellular parasites, viruses expertly modify cellular processes to facilitate their replication and spread, often by encoding genes that mimic the functions of cellular proteins while lacking regulatory features that modify their activity. We show that the human cytomegalovirus UL97 protein has activities similar to cellular cyclin–cyclin-dependent kinase (CDK) complexes. UL97 phosphorylated and inactivated the retinoblastoma tumor suppressor, stimulated cell cycle progression in mammalian cells, and rescued proliferation of
Saccharomyces cerevisiae
lacking CDK activity. UL97 is not inhibited by the CDK inhibitor p21 and lacks amino acid residues conserved in the CDKs that permit the attenuation of kinase activity. Thus, UL97 represents a functional ortholog of cellular CDKs that is immune from normal CDK control mechanisms.
Discovery of a Cytokine and Its Receptor by Functional Screening of the Extracellular Proteome
Authors: Lin Haishan , Lee Ernestine , Hestir Kevin , Leo Cindy , Huang Minmei , Bosch Elizabeth , Halenbeck Robert , Wu Ge , Zhou Aileen , Behrens Dirk , Hollenbaugh Diane , Linnemann Thomas , Qin Minmin , Wong Justin , Chu Keting , Doberstein Stephen K. , Williams Lewis T.
Published: May 2008

To understand the system of secreted proteins and receptors involved in cell-cell signaling, we produced a comprehensive set of recombinant secreted proteins and the extracellular domains of transmembrane proteins, which constitute most of the protein components of the extracellular space. Each protein was tested in a suite of assays that measured metabolic, growth, or transcriptional responses in diverse cell types. The pattern of responses across assays was analyzed for the degree of functional selectivity of each protein. One of the highly selective proteins was a previously undescribed ligand, designated interleukin-34 (IL-34), which stimulates monocyte viability but does not affect responses in a wide spectrum of other assays. In a separate functional screen, we used a collection of extracellular domains of transmembrane proteins to discover the receptor for IL-34, which was a known cytokine receptor, colony-stimulating factor 1 (also called macrophage colony-stimulating factor) receptor. This systematic approach is thus useful for discovering new ligands and receptors and assessing the functional selectivity of extracellular regulatory proteins.
Spatial Regulators for Bacterial Cell Division Self-Organize into Surface Waves in Vitro
Authors: Loose Martin , Fischer-Friedrich Elisabeth , Ries Jonas , Kruse Karsten , Schwille Petra
Published: May 2008

In the bacterium
Escherichia coli
, the Min proteins oscillate between the cell poles to select the cell center as division site. This dynamic pattern has been proposed to arise by self-organization of these proteins, and several models have suggested a reaction-diffusion type mechanism. Here, we found that the Min proteins spontaneously formed planar surface waves on a flat membrane in vitro. The formation and maintenance of these patterns, which extended for hundreds of micrometers, required adenosine 5′-triphosphate (ATP), and they persisted for hours. We present a reaction-diffusion model of the MinD and MinE dynamics that accounts for our experimental observations and also captures the in vivo oscillations.
Architecture of a Charge-Transfer State Regulating Light Harvesting in a Plant Antenna Protein
Authors: Ahn Tae Kyu , Avenson Thomas J. , Ballottari Matteo , Cheng Yuan-Chung , Niyogi Krishna K. , Bassi Roberto , Fleming Graham R.
Published: May 2008

Energy-dependent quenching of excess absorbed light energy (qE) is a vital mechanism for regulating photosynthetic light harvesting in higher plants. All of the physiological characteristics of qE have been positively correlated with charge transfer between coupled chlorophyll and zeaxanthin molecules in the light-harvesting antenna of photosystem II (PSII). We found evidence for charge-transfer quenching in all three of the individual minor antenna complexes of PSII (CP29, CP26, and CP24), and we conclude that charge-transfer quenching in CP29 involves a delocalized state of an excitonically coupled chlorophyll dimer. We propose that reversible conformational changes in CP29 can “tune” the electronic coupling between the chlorophylls in this dimer, thereby modulating the energy of the chlorophyll-zeaxanthin charge-transfer state and switching on and off the charge-transfer quenching during qE.
Recombinant Group BStreptococcusAlpha-Like Protein 3 Is an Effective Immunogen and Carrier Protein
Authors: Yang Hsiao-Hui , Mascuch Samantha J. , Madoff Lawrence C. , Paoletti Lawrence C.
Published: May 2008

Conjugate vaccines against pathogens of multiple serotypes are optimized when all components induce functional antibody, resulting in broadened coverage. While most clinical studies of vaccines against group BStreptococcus(GBS) have evaluated conjugates composed of capsular polysaccharide (CPS) coupled to tetanus toxoid, conjugates prepared with GBS proteins as carriers have also been efficacious in animals. Here, we report that recombinant GBS alpha-like protein 3 (rAlp3) is both a strong immunogen and a viable carrier protein for type III CPS. The type III CPS-specific immunoglobulin G (IgG) titer rose from <100 to 64,000 among mice that received type III CPS coupled to rAlp3 (III-rAlp3) compared with an absence of a specific response among mice that received an uncoupled mixture. Most (94%) newborn pups born to III-rAlp-vaccinated dams survived challenge with viable type III GBS, compared with 43% survival among those born to dams that received the uncoupled mixture (P< 0.0001). A tricomponent conjugate of type III CPS, rAlp3, and a GBS recombinant beta C protein lacking its IgA binding site (III-rAlp3-rBCPΔIgA) provided protection against a serotype III strain and a serotype Ia strain bearing beta C protein. High-titered anti-rAlp3 rabbit serum opsonized Alp3-containing strains of two GBS serotypes (types V and VIII) and invasive type III strains bearing the cross-reactive Rib protein for in vitro killing by human peripheral blood leukocytes. Thus, the potential exists for the inclusion of rAlp3 in a GBS vaccine formulated to provide multiserotype coverage.
Immunological Evidence for Functional Rather than Structural Mimicry by aShigella flexneriY Polysaccharide-Mimetic Peptide
Authors: Borrelli Silvia , Hossany Rehana B. , Pinto B. Mario
Published: May 2008

An approach to vaccine design is the use of molecules that mimic the immunogenic element of interest. In this context, the interaction of MDWNMHAA, a peptide mimic of theShigella flexneriY O polysaccharide (PS), with an anti-carbohydrate monoclonal antibody, as studied previously by X-ray crystallography, suggested the presence of functional rather than structural mimicry and a bound peptide conformation that was not represented significantly in the free-ligand ensemble. The antibody response elicited by an MDWNMHAA-carrier protein (tetanus toxoid [TT]) conjugate has now been investigated in BALB/c mice. The mice were immunized following a homologous prime/boost strategy using MDWNMHAA-TT as the immunogen. The mice showed anti-peptide antibody (immunoglobulin G [IgG]) titers that increased after being boosted. High anti-lipopolysaccharide (LPS) (IgG) titers were observed after the last boost. A faster immune response, with cross-reactive titers, was observed with a peptide conjugate with 30% more copies of the peptide. The binding of anti-peptide polyclonal antibodies to LPS could be inhibited by LPS, PS, MDWNMHAA, and MDWNMHAA-bovine serum albumin, as assessed by inhibition enzyme-linked immunosorbent assay. Conversely, mice immunized with PS-TT showed IgG anti-peptide titers. These data demonstrate the cross-reactivity of the antibody response and support the hypothesis that functional, as opposed to structural, mimicry of theS. flexneriY O PS by MDWNMHAA or the underrepresentation of the bound ligand conformation in the free-ligand ensemble does not compromise immunological cross-reactivity. Prime/boost strategies were performed with a heterologous boost of PS-TT or MDWNMHAA-TT. They led to high anti-LPS titers after only three injections, suggesting alternatives to improve the immunogenicity of the carbohydrate-mimetic peptide and confirming the antigenic mimicry.
Quadruplex PCR for Simultaneous Detection of Serotype, Biotype, Toxigenic Potential, and Central Regulating Factor of
Vibrio cholerae
Authors: Khuntia Hemant Kumar , Pal Bibhuti Bhusan , Chhotray Guru Prasada
Published: May 2008

A quadruplex PCR was developed for the simultaneous detection of genes specific for
Vibrio cholerae
O1 and/or O139 serogroup (
wbe
and/or
wbf
), cholera toxin A subunit (
ctxA
), toxin-coregulated pilus (
tcpA
), and central regulating protein ToxR (
toxR
) in a single tube reaction. This is a simple, rapid, and accurate approach for the detection of toxigenic
V. cholerae
O1 and/or O139 and can prevent the rapid spread of the disease by early detection.
Rapid Identification of Penicillin and Macrolide Resistance Genes and Simultaneous Quantification of
Streptococcus pneumoniae
in Purulent Sputum Samples by Use of a Novel Real-Time Multiplex PCR Assay
Authors: Fukushima Kazuko Y. , Yanagihara Katsunori , Hirakata Yoichi , Sugahara Kazuyuki , Morinaga Yoshitomo , Kohno Shigeru , Kamihira Shimeru
Published: May 2008

We evaluated a real-time quantitative PCR combined with a multiplex PCR assay for the quantification of
Streptococcus pneumoniae
and the simultaneous detection of drug-resistant genes by gel-based PCR, using purulent sputum samples. This assay correctly quantified
S. pneumoniae
and identified their penicillin and erythromycin susceptibilities directly from samples within 3 h.
Highly Sensitive Methods Based on Seminested Real-Time Reverse Transcription-PCR for Quantitation of Human Immunodeficiency Virus Type 1 Unspliced and Multiply Spliced RNA and Proviral DNA
Authors: Pasternak Alexander O. , Adema Karen W. , Bakker Margreet , Jurriaans Suzanne , Berkhout Ben , Cornelissen Marion , Lukashov Vladimir V.
Published: May 2008

The effectiveness of highly active antiretroviral therapy (HAART), the standard of care for the treatment of human immunodeficiency virus type 1 (HIV-1) infection, is assessed by measuring the viral RNA load in plasma. A patient is considered to be successfully treated when the HIV-1 load in plasma stays below the detection limit of commercial assays. However, virus replication and evolution do continue in patients under HAART, which may eventually result in the development of drug-resistant HIV-1 strains and therapy failure. To monitor this low-level virus replication in peripheral blood mononuclear cells (PBMC), sensitive methods are required to measure HIV-1 molecular markers. We report the development of highly sensitive methods for the quantitation of unspliced and multiply spliced HIV-1 RNA and proviral DNA in PBMC. The methods are based on innovative seminested real-time reverse transcription-PCR (RT-PCR) that combines the accuracy and precision of real-time PCR and the sensitivity of nested PCR. We show that the newly developed methods are superior to the conventional single-step real-time RT-PCR in their sensitivity, accuracy, dynamic range, and the power of quantitative detection of HIV-1 RNA and DNA in clinical samples. These easy-to-perform methods can be widely used in research, including clinical studies, to monitor intracellular processes of virus replication.
Evaluation of Two Commercial Systems for Automated Processing, Reading, and Interpretation of Lyme Borreliosis Western Blots
Authors: Binnicker M. J. , Jespersen D. J. , Harring J. A. , Rollins L. O. , Bryant S. C. , Beito E. M.
Published: May 2008

The diagnosis of Lyme borreliosis (LB) is commonly made by serologic testing with Western blot (WB) analysis serving as an important supplemental assay. Although specific, the interpretation of WBs for diagnosis of LB (i.e., Lyme WBs) is subjective, with considerable variability in results. In addition, the processing, reading, and interpretation of Lyme WBs are laborious and time-consuming procedures. With the need for rapid processing and more objective interpretation of Lyme WBs, we evaluated the performances of two automated interpretive systems, TrinBlot/BLOTrix (Trinity Biotech, Carlsbad, CA) and BeeBlot/ViraScan (Viramed Biotech AG, Munich, Germany), using 518 serum specimens submitted to our laboratory for Lyme WB analysis. The results of routine testing with visual interpretation were compared to those obtained by BLOTrix analysis of MarBlot immunoglobulin M (IgM) and IgG and by ViraScan analysis of ViraBlot and ViraStripe IgM and IgG assays. BLOTrix analysis demonstrated an agreement of 84.7% for IgM and 87.3% for IgG compared to visual reading and interpretation. ViraScan analysis of the ViraBlot assays demonstrated agreements of 85.7% for IgM and 94.2% for IgG, while ViraScan analysis of the ViraStripe IgM and IgG assays showed agreements of 87.1 and 93.1%, respectively. Testing by the automated systems yielded an average time savings of 64 min/run compared to processing, reading, and interpretation by our current procedure. Our findings demonstrated that automated processing and interpretive systems yield results comparable to those of visual interpretation, while reducing the subjectivity and time required for Lyme WB analysis.
Molecular Characterization of
Staphylococcus aureus
Bloodstream Isolates Collected in a Dutch University Hospital between 1999 and 2006
Authors: Nulens Eric , Stobberingh Ellen E. , van Dessel Helke , Sebastian Silvie , van Tiel Frank H. , Beisser Patrick S. , Deurenberg Ruud H.
Published: May 2008

We observed that, between 1999 and 2006, up to 50% of the methicillin-susceptible
Staphylococcus aureus
(MSSA) bloodstream isolates in our hospital had a genetic background common to endemic methicillin-resistant
S. aureus
clones (clonal complex 5 [CC5], CC8, CC22, CC30, and CC45). Furthermore, several successful MSSA lineages, such as CC7 and CC15, were observed.
Novel Subgenotypes of Hepatitis B Virus Genotypes C and D in Papua, Indonesia
Authors: Lusida Maria Inge , Nugrahaputra Victor Eka , Soetjipto  , Handajani Retno , Nagano-Fujii Motoko , Sasayama Mikiko , Utsumi Takako , Hotta Hak
Published: May 2008

Eight genotypes (A to H) and nine subtypes (
adw2
,
adw4
,
ayw1
,
ayw2
,
ayw3
,
ayw4
,
adrq
+,
adrq
−, and
ayr
) of hepatitis B virus (HBV) have been identified worldwide. They appear to be associated with geographical distribution, virological characteristics, and possibly clinical outcomes. We performed sequence analysis of part of the S gene and the entire precore/core gene of HBV isolates obtained from HBsAg-positive blood donors in Papua Province, Indonesia. Phylogenetic analysis of the S gene sequences revealed that 23 (85.2%) of the 27 HBV isolates tested belonged to genotype C (HBV/C) and 2 (7.4%) each to HBV/B and HBV/D. Interestingly, 19 (82.6%) of the 23 isolates of HBV/C clustered in a branch that was distinct from the previously reported subgenotypes C1 to C5 (HBV/C1 to HBV/C5). Similarly, two isolates of HBV/D clustered in a branch distinct from the reported subgenotypes HBV/D1 to HBV/D5. Phylogenetic analysis of the entire precore/core gene confirmed the consistent presence of the distinct branches in HBV/C and HBV/D. We therefore propose novel subgenotypes designated HBV/C6 and HBV/D6. The majority of HBV/C6 isolates in Papua had alanine at positions 159 and 177 (A159/A177) in the HBsAg. A159/A177 is different from the determinants for
adrq
+ (A159/V177), found throughout Asia, and
adrq
− (V159/A177), found in New Caledonia and Polynesia, possibly representing a unique antigenic group (provisionally referred to as
adrq
indeterminate). In conclusion, we have identified two novel HBV subgenotypes, HBV/C6 and HBV/D6, the first of which is the most prevalent subgenotype of HBV in Papua, Indonesia.
A Live Attenuated Severe Acute Respiratory Syndrome Coronavirus Is Immunogenic and Efficacious in Golden Syrian Hamsters
Authors: Lamirande Elaine W. , DeDiego Marta L. , Roberts Anjeanette , Jackson Jadon P. , Alvarez Enrique , Sheahan Tim , Shieh Wun-Ju , Zaki Sherif R. , Baric Ralph , Enjuanes Luis , Subbarao Kanta
Published: May 2008

The immunogenicity and protective efficacy of a live attenuated vaccine consisting of a recombinant severe acute respiratory syndrome (SARS) coronavirus lacking the E gene (rSARS-CoV-ΔE) were studied using hamsters. Hamsters immunized with rSARS-CoV-ΔE developed high serum-neutralizing antibody titers and were protected from replication of homologous (SARS-CoV Urbani) and heterologous (GD03) SARS-CoV in the upper and lower respiratory tract. rSARS-CoV-ΔE-immunized hamsters remained active following wild-type virus challenge, while mock-immunized hamsters displayed decreased activity. Despite being attenuated in replication in the respiratory tract, rSARS-CoV-ΔE is an immunogenic and efficacious vaccine in hamsters.
Genotypic Features of Lentivirus Transgenic Mice
Authors: Sauvain Marc-Olivier , Dorr Alexander P. , Stevenson Brian , Quazzola Alexandra , Naef Félix , Wiznerowicz Maciej , Schütz Frédéric , Jongeneel Victor , Duboule Denis , Spitz François , Trono Didier
Published: May 2008

Lentivector-mediated transgenesis is increasingly used, whether for basic studies as an alternative to pronuclear injection of naked DNA or to test candidate gene therapy vectors. In an effort to characterize the genetic features of this approach, we first measured the frequency of germ line transmission of individual proviruses established by infection of fertilized mouse oocytes. Seventy integrants from 11 founder (G0) mice were passed to 111 first generation (G1) pups, for a total of 255 events corresponding to an average rate of transmission of 44%. This implies that integration had most often occurred at the one- or two-cell stage and that the degree of genotypic mosaicism in G0 mice obtained through this approach is generally minimal. Transmission analysis of eight individual proviruses in 13 G2 mice obtained by a G0-G1 cross revealed only 8% of proviral homozygosity, significantly below the 25% expected from purely Mendelian transmission, suggesting counter-selection due to interference with the functions of targeted loci. Mapping of 239 proviral integration sites in 49 founder animals revealed that about 60% resided within annotated genes, with a marked tendency for clustering in the middle of the transcribed region, and that integration was not influenced by the transcriptional orientation. Transcript levels of a set of arbitrarily chosen target genes were significantly higher in two-cell embryos than in embryonic stem cells or adult somatic cells, suggesting that, as previously noted in other settings, lentiviral vectors integrate preferentially into regions of the genome that are transcriptionally active or poised for activation.
Role of Initiating Nucleoside Triphosphate Concentrations in the Regulation of Influenza Virus Replication and Transcription
Authors: Vreede Frank T. , Gifford Hugh , Brownlee George G.
Published: May 2008

The mechanisms regulating the synthesis of mRNA, cRNA, and viral genomic RNA (vRNA) by the influenza A virus RNA-dependent RNA polymerase are not fully understood. Previous studies in our laboratory have shown that virion-derived viral ribonucleoprotein complexes synthesize both mRNA and cRNA in vitro and early in the infection cycle in vivo. Our continued studies showed that de novo synthesis of cRNA in vitro is more sensitive to the concentrations of ATP, CTP, and GTP than capped-primer-dependent synthesis of mRNA. Using rescued recombinant influenza A/WSN/33 viruses, we now demonstrate that the 3′-terminal sequence of the vRNA promoter dictates the requirement for a high nucleoside triphosphate (NTP) concentration during de novo-initiated replication to cRNA, whereas this is not the case for the extension of capped primers during transcription to mRNA. In contrast to some other viral polymerases, for which only the initiating NTP is required at high concentrations, influenza virus polymerase requires high concentrations of the first three NTPs. In addition, we show that base pair mutations in the vRNA promoter can lead to nontemplated dead-end mutations during replication to cRNA in vivo. Based on our observations, we propose a new model for the de novo initiation of influenza virus replication.
Development of Cell Lines That Provide Tightly Controlled Temporal Translation of the Human Cytomegalovirus IE2 Proteins for Complementation and Functional Analyses of Growth-Impaired and Nonviable IE2 Mutant Viruses
Authors: Sanders Rebecca L. , Clark Charles L. , Morello Christopher S. , Spector Deborah H.
Published: May 2008

The human cytomegalovirus (HCMV) IE2 86 protein is essential for viral replication. Two other proteins, IE2 60 and IE2 40, which arise from the C-terminal half of IE2 86, are important for later stages of the infection. Functional analyses of IE2 86 in the context of the infection have utilized bacterial artificial chromosomes as vectors to generate mutant viruses. One limitation is that many mutations result in debilitated or nonviable viruses. Here, we describe a novel system that allows tightly controlled temporal expression of the IE2 proteins and provides complementation of both growth-impaired and nonviable IE2 mutant viruses. The strategy involves creation of cell lines with separate lentiviruses expressing a bicistronic RNA with a selectable marker as the first open reading frame (ORF) and IE2 86, IE2 60, or IE2 40 as the second ORF. Induction of expression of the IE2 proteins occurs only following DNA recombination events mediated by Cre and FLP recombinases that delete the first ORF. HCMV encodes Cre and FLP, which are expressed at immediate-early (for IE2 86) and early-late (for IE2 40 and IE2 60) times, respectively. We show that the presence of full-length IE2 86 alone provides some complementation for virus production, but the correct temporal expression of IE2 86 and IE2 40 together has the most beneficial effect for early-late gene expression and synthesis of infectious virus. This approach for inducible protein translation can be used for complementation of other mutations as well as controlled expression of toxic cellular and microbial proteins.
Cdc34p Ubiquitin-Conjugating Enzyme Is a Component of the Tombusvirus Replicase Complex and Ubiquitinates p33 Replication Protein
Authors: Li Zhenghe , Barajas Daniel , Panavas Tadas , Herbst David A. , Nagy Peter D.
Published: May 2008

To identify host proteins interacting with
Tomato bushy stunt virus
(TBSV) replication proteins in a genome-wide scale, we have used a yeast (
Saccharomyces cerevisiae
) proteome microarray carrying 4,088 purified proteins. This approach led to the identification of 58 yeast proteins that interacted with p33 replication protein. The identified host proteins included protein chaperones, ubiquitin-associated proteins, translation factors, RNA-modifying enzymes, and other proteins with yet-unknown functions. We confirmed that 19 of the identified host proteins bound to p33 in vitro or in a split-ubiquitin-based two-hybrid assay. Further analysis of Cdc34p E2 ubiquitin-conjugating enzyme, which is one of the host proteins interacting with p33, revealed that Cdc34p is a novel component of the purified viral replicase. Downregulation of Cdc34p expression in yeast, which supports replication of a TBSV replicon RNA (repRNA), reduced repRNA accumulation and the activity of the tombusvirus replicase by up to fivefold. Overexpression of wild-type Cdc34p, but not that of an E2-defective mutant of Cdc34p, increased repRNA accumulation, suggesting a significant role for the ubiquitin-conjugating enzyme function of Cdc34p in TBSV replication. Also, Cdc34p was able to ubiquitinate p33 in vitro. In addition, we have shown that p33 becomes ubiquitinated in vivo. We propose that ubiquitination of p33 likely alters its function or affects the recruitment of host factors during TBSV replication.
Effect of graded bicycle seat pressure on perineal compression: a magnetic resonance imaging analysis: Figure 1
Authors: Bressel Eadric , Parker Dan
Published: May 2008

Objective

To identify a seat pressure threshold that corresponds to significant compression of the perineum.

Design

Quasiexperimental crossover.

Setting

Hospital radiology department.

Participants

Convenience sample of six male recreational cyclists.

Intervention

Compression of the corpus spongiosum and corpora cavernosa of the perineum without bicycle seat pressure (0%) and at 10%, 40% and 80% of the mean bicycling pressure was assessed using magnetic resonance imaging. Seat pressure was applied using a custom loading device.

Outcome Measure

Measure Diameter measurements of the cavernous spaces at the point of peak compression.

Results

The mean diameter values for the corpus spongiosum in the unloaded and 10% of load conditions were 105% and 172% greater than the 40% and 80% loaded conditions, respectively (p=0.002–0.004). The corpora cavernosa values displayed a similar trend.

Conclusions

Substantial compression of the perineal cavernous spaces may occur with total seat pressure values of less than 40% of typical seat loads.
Shoulder pain in elite swimmers: primarily due to swim-volume-induced supraspinatus tendinopathy
Authors: Sein Mya Lay , Walton Judie , Linklater James , Appleyard Richard , Kirkbride Brent , Kuah Donald , Murrell George A C
Published: May 2008

Background/hypothesis

Shoulder pain in elite swimmers is common, and its pathogenesis is uncertain.

Hypothesis/study design

The authors used a crosssectional study design to test Jobe’s hypothesis that repetitive forceful swimming leads to shoulder laxity, which in turn leads to impingement pain.

Methods

Eighty young elite swimmers (13–25 years of age) completed questionnaires on their swimming training, pain and shoulder function. They were given a standardised clinical shoulder examination, and tested for glenohumeral joint laxity using a non-invasive electronic laxometer. 52/80 swimmers also attended for shoulder MRI.

Results

73/80 (91%) swimmers reported shoulder pain. Most (84%) had a positive impingement sign, and 69% of those examined with MRI had supraspinatus tendinopathy. The impingement sign and MRIdetermined supraspinatus tendinopathy correlated strongly (rs=0.49, p<0.00001). Increased tendon thickness correlated with supraspinatus tendinopathy (rs=0.37, p<0.01). Laxity correlated weakly with impingement pain (rs=0.23, p<0.05) and was not associated with supraspinatus tendinopathy (rs=0.14, p=0.32). The number of hours swum/week (rs=0.39, p<0.005) and weekly mileage (rs=0.34, p=0.01) both correlated significantly with supraspinatus tendinopathy. Swimming stroke preference did not.

Conclusions

These data indicate: (1) supraspinatus tendinopathy is the major cause of shoulder pain in elite swimmers; (2) this tendinopathy is induced by large amounts of swimming training; and (3) shoulder laxity per se has only a minimal association with shoulder impingement in elite swimmers. These findings are consistent with animal and tissue culture findings which support an alternate hypothesis: the intensity and duration of load to tendon fibres and cells cause tendinopathy, impingement and shoulder pain.
Sequence characterization of the putatively sex-linked Ssu72-like locus in willow and its homologue in poplarThis note is one of a selection of papers published in the Special Issue on Poplar Research in Canada.
Authors: Temmel Nyssa A. , Rai Hardeep S. , Cronk Quentin C.B.
Published: May 2008

Previous studies have identified a sequence characterized amplified region (SCAR) marker in Salix viminalis L. that appears to segregate with gender. To characterize this marker, we tested these SCAR primers in poplar ( Populus trichocarpa Torr. & A. Gray) and 12 species of willow ( Salix ). Although amplification was inconsistent with respect to species and gender, products were obtained in four willow species (but not in poplar). The resulting sequences show that the SCAR consists of (i) a length variable purine-rich repeat region and (ii) a region highly conserved between species. The conserved region has an apparent homologue in the poplar genome, where it corresponds to the putative promoter region of an Ssu72-like gene (involved in transcriptional start site regulation) on chromosome XV. We used the poplar genome sequence to design gene-anchored primers that consistently amplify this region and part of the Ssu72-like coding region in willows as well as poplars, irrespective of species and gender. The gene-anchored primers amplify a region that, while conserved, has numerous single feature polymorphisms (SFPs) both within and between species. This region could thus be used for population and phylogenetic studies.
The interaction between nitrogen source, soil pH, and drought in the growth and physiology of three poplar clonesThis article is one of a selection of papers published in the Special Issue on Poplar Research in Canada.
Authors: DesRochers Annie , van den Driessche R. , Thomas Barb R.
Published: May 2008

The objective of this study was to understand how nitrogen (N) source, soil pH, and drought interact in the growth and water-use efficiency of poplars. Rooted cuttings from two hybrid poplar clones, 24 (Walker) ( Populus deltoides × Populus ×petrowskyana ), and 794 (Brooks 6) (Populus deltoides × Populus ×petrowskyana), and one native balsam poplar clone 1004 ( Populus balsamifera L.), were grown in a greenhouse for a period of 8 weeks. Trees received a combination of two N sources (ammonium sulphate [(NH4)2SO4] and calcium nitrate [Ca(NO3)2]), two soil pHs (5 and 7), and two drought levels (85% and 40% field capacity, as determined by mass). Trees had reduced growth, net photosynthesis, and stomatal conductance with NO3– fertilization, and these effects were accentuated at soil pH  7. The drought treatment only slightly reduced growth and rarely interacted with N source or soil pH. Drought also did not affect instantaneous measurements of water-use efficiency, but it increased δ13C by an average of 0.55‰. The poplar clones used in this study appear to be better adapted to take up N from NH4+ than from NO3–-N sources.
Molecular characterization of GATA/GACA microsatellite repeats in tomato
Authors: Vosman B. , Arens P.
Published: May 2008

Microsatellite repeats like GATA or GACA display a degree of variability that allows their use in cultivar identification. Southern hybridization with oligonucleotide probes complementary to these microsatellites were used for the detection of polymorphisms. To understand the molecular structure of the detected DNA, fragments hybridizing to GATA and GACA probes were cloned and sequenced. In the four clones analyzed, repeats of GATA and GACA were found intertwined. The GATA and GACA arrays were not perfect but were heavily degenerated, in that they contained many tetranucleotides that might have been derived by a single point mutation from GATA or GACA. Some of these derived sequences, like GGTA and GGAT, were present as relatively long stretches that also contained some point mutations. This supports the hypothesis that long stretches of repeats are stabilized by the accumulation of point mutations. Analysis of the flanking sequences of the fragments obtained with the GACA probe showed that one of them was homologous to a Lilium henryi retrotransposon and the other to a sequence upstream of a potato patatin gene. The two fragments obtained using the GATA probe were flanked by DNA that had no homology to any known sequence but they were highly homologous to each other. This DNA was frequently associated with GATA elements and was present in the tomato genome in approximately 4300 copies. The function of this new class of repetitive DNA, here termed U30, is presently unknown.Key words: simple sequence repeats, Lycopersicon esculentum, cultivar identification, repetitive DNA.
Combined RAPD and RFLP molecular linkage map of asparagus
Authors: Jiang C. , Lewis M. E. , Sink K. C.
Published: May 2008

Two linkage maps of asparagus (Asparagus officinalis L.) were constructed using a double pseudotestcross mapping strategy with restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), and allozymes as markers in a population generated from crossing MW25 × A19, two heterozygous parents. All data were inverted and combined with the natural data to detect linkages in repulsion phase. Two sets of data, one for each parent, were formed according to the inheritance patterns of the markers. The maternal MW25 map has a total of 163 marker loci placed in 13 linkage groups covering 1281 cM, with an average and a maximum distance between adjacent loci of 7.9 and 29 cM, respectively. The paternal A19 map has 183 marker loci covering 1324 cM in 9 linkage groups, with an average and a maximum distance between two adjacent loci of 7.7 and 29 cM, respectively. Six multiallelic RFLPs segregating in the pattern a/c × b/c and eight heterozygous loci (four RAPDs, and four RFLPs segregating in the pattern a/b × a/b (HZ loci)) were common to both maps. These 14 loci were used as bridges to align homologous groups between the two maps. In this case, RFLPs were more frequent and informative than RAPDs. Nine linkage groups in the MW25 map were homologous to six groups in the A19 map. In two cases, two or more bridge loci were common to a group; thus, the orientation of homologous linkage groups was also determined. In four other cases, only one locus was common to the two homologous groups and the orientation was unknown. Mdh, four RFLPs, and 14 RAPDs were assigned to chromosome L5, which also has the sex locus M.Key words: asparagus, bridge loci, pseudotestcross, RAPD, RFLP, sex expression.
Involvement of knob heterochromatin in mitotic abnormalities in germinating aged seeds of maize
Authors: Fluminhan A. , Kameya T.
Published: May 2008

Seeds of three inbred lines of maize, with contrasting numbers of heterochromatic knobs and stored under two different ageing treatments, were analyzed for the occurrence of abnormalities at mitotic anaphase in root meristems 3, 7, 21,42, and 56 days after germination, and in root meristems of their freshly harvested selfed progeny. The largest category of detectable aberrations involved breakage of knobbed chromosome arms. We have obtained evidence that knob heterochromatin plays a central role in the origin of primary chromosome bridges. The initial event responsible for the occurrence of breakages and lagging chromosomes was characterized by the nondisjunction of newly replicated sister chromatids, which was observed to occur preferentially at the knob level. Such configurations, and all the other types of abnormalities (as for example, lagging chromosomes, typical chromosome bridges, fragments, and micronuclei), were observed at decreasing frequencies throughout root growth. Nevertheless, we have detected the occurrence of breakage–fusion–bridge cycles that were initiated by broken chromosomes. The relationship between late-replicating DNA in maize knob heterochromatin and the vulnerability of such regions to breakage is discussed. Our observations suggest a similarity between the mechanisms involved or associated with the origin of the described abnormalities and those reported to occur in cultured maize cells.Key words: Zea mays L., seed ageing, aberrations in chromosome structure, heterochromatic knobs, chromosome behavior.
Chloroplast DNA variability in wild and cultivated rice (Oryzaspp.) revealed by polymorphic chloroplast simple sequence repeats
Authors: Provan J. , Corbett G. , Powell W. , McNicol J. W.
Published: May 2008

Short mononucleotide repeats analogous to nuclear microsatellites or simple sequence repeats (SSRs) have been identified in chloroplast genomes. Primers flanking mononucleotide repeats in the fully sequenced rice chloroplast genome have been used in conjunction with PCR to amplify genomic DNA from 42 wild rice accessions. The amplification products exhibited length polymorphism, which allowed the levels of chloroplast variability detected to be quantified. Seven primer pairs that amplified products from different regions of the rice chloroplast were used, five of which also amplified polymorphic products in cultivated rice (Oryza sativa). Diversity values ranged from 0.5224 ± 0.0845 (SE) to 0.8298 ± 0.0085 in the wild accessions, which was higher than that detected in the O. sativa accessions. Both intra- and inter-specific polymorphism was detected, and the extent of chloroplast genomic differentiation based on chloroplast simple sequence repeat (cpSSR) assays was quantified using the RSTstatistic. Primers designed to amplify cpSSRs in O. sativa can also be used to generate polymorphic chloroplast markers in related taxa. The potential of using cpSSR to trace the origin of rice polyploid species is discussed.Key words: rice, chloroplast, simple sequence repeat, microsatellites.
Dynamics of Sau3A in situ digestion of human chromosomes analyzed with computerized imaging
Authors: Buño Ismael , López-Fernández Carmen , Gosálvez Jaime , Díez-Martín José Luis
Published: May 2008

The differential DNA removal obtained after in situ digestion of human metaphase chromosomes with the restriction endonuclease Sau3A was analyzed on complete and partially digested nuclei using computerized imaging. The results obtained permit the discrimination of Sau3A-resistant chromosome regions (pericentromeric constitutive heterochromatin on chromosomes 3 and 9) from those partially digested (Yq12 and pericentromeric heterochromatin on other chromosomes), according to the digestion dynamics deduced from the grey intensity profile along each chromosome. This approach permits an accurate labelling of chromosome markers for the identification of genomes from different individuals. This is of special interest for the analysis of the chimeric status found in patients after allogenic bone marrow transplantation.Key words: human cytogenetics, restriction endonucleases, in situ digestion, image processing and analysis.
Atomic force microscope imaging of chromosome structure during G-banding treatments
Authors: Musio Antonio , Sbrana Isabella , Mariani Tullio , Frediani Carlo , Ascoli Cesare
Published: May 2008

Surface topography of human chromosomes was examined by atomic force microscopy during treatments for G-banding. Trypsin treatment resulted in a structural modification in the chromatin. Subsequent Giemsa staining caused a general swelling of the chromosomal surface that was greater in the areas of G-band positive regions. By means of a quantitative evaluation method we showed that the G-banding process produces a 10-fold enhancement of a pre-existing pattern of chromatin between G-band positive and G-band negative regions on mitotic chromosomes.Key words: atomic force microscopy, human chromosomes, G-banding, trypsin treatment, Giemsa stain.
A white eye color mutant in the tsetse fly Glossina morsitans submotsitans Newstead (Diptera: Glossinidae)
Authors: Challoner C. M. , Gooding R. H.
Published: May 2008

A spontaneous mutation in Glossina morsitans submorsitans Newstead is described. The mutant, designated wht, has white compound eyes but the ocelli and testes have normal coloration. Mutants have lower than normal amounts of xanthommatin and pteridines in their heads. The lesion occurs late in the tryptophan to xanthommatin pathway, in the storage of xanthommatin in the compound eyes, or, most likely, in the transport of precursors into the compound eyes. The locus wht is on the X chromosome.Key words: tsetse, Glossina morsitans submorsitans, wht mutant, tryptophan metabolism.