bluuebunny/crossref_metadata_2025 huggingface dataset:
The relativistic Ampère–Maxwell law in integral form
Authors: Monsivais G.
Published: August 2004

We discuss an extension of the integral form of the Ampère–Maxwell law for surfaces moving with relativistic velocities in the presence of electromagnetic fields, including when different parts of the surface move with arbitrary accelerated and deformed motion. We also discuss the case of Faraday’s law and compare our result to an expression of the same type derived by Gelman. These general formulations are necessary for theoretical consistency, but are absent from textbooks.
Wave packet propagation into a negative index medium
Authors: Huang Xiaobiao , Schaich W. L.
Published: August 2004

We examine the phenomenon of negative refraction using wave packet propagation. The behavior of a single plane wave incident from vacuum onto a flat surface of a medium with a negative index of refraction is solved analytically. The solution requires matching electromagnetic field components across the interface and produces a reflected and a refracted wave in addition to the incident wave. We form by numerical integration a (Gaussian-weighted) linear combination of these solutions. Pictures and videos that illustrate the resulting wave packet motion are presented for a variety of parameter choices.
Increased Nuclear NAD Biosynthesis and SIRT1 Activation Prevent Axonal Degeneration
Authors: Araki Toshiyuki , Sasaki Yo , Milbrandt Jeffrey
Published: August 2004

Axonal degeneration is an active program of self-destruction that is observed in many physiological and pathological settings. In Wallerian degeneration slow (

wld
s

) mice, Wallerian degeneration in response to axonal injury is delayed because of a mutation that results in overexpression of a chimeric protein (Wld
s
) composed of the ubiquitin assembly protein Ufd2a and the nicotinamide adenine dinucleotide (NAD) biosynthetic enzyme Nmnat1. We demonstrate that increased Nmnat activity is responsible for the axon-sparing activity of the Wld
s
protein. Furthermore, we demonstrate that SIRT1, a mammalian ortholog of Sir2, is the downstream effector of increased Nmnat activity that leads to axonal protection. These findings suggest that novel therapeutic strategies directed at increasing the supply of NAD and/or Sir2 activation may be effective for treatment of diseases characterized by axonopathy and neurodegeneration.
Drug Seeking Becomes Compulsive After Prolonged Cocaine Self-Administration
Authors: Vanderschuren Louk J. M. J. , Everitt Barry J.
Published: August 2004

Compulsive drug use in the face of adverse consequences is a hallmark feature of addiction, yet there is little preclinical evidence demonstrating the actual progression from casual to compulsive drug use. Presentation of an aversive conditioned stimulus suppressed drug seeking in rats with limited cocaine self-administration experience, but no longer did so after an extended cocaine-taking history. In contrast, after equivalent extended sucrose experience, sucrose seeking was still suppressed by an aversive conditioned stimulus. Persistent cocaine seeking in the presence of signals of environmental adversity after a prolonged cocaine-taking history was not due to impaired fear conditioning, nor to an increase in the incentive value of cocaine, and may reflect the establishment of compulsive behavior.
Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy
Authors: Huisken Jan , Swoger Jim , Del Bene Filippo , Wittbrodt Joachim , Stelzer Ernst H. K.
Published: August 2004

Large, living biological specimens present challenges to existing optical imaging techniques because of their absorptive and scattering properties. We developed selective plane illumination microscopy (SPIM) to generate multidimensional images of samples up to a few millimeters in size. The system combines two-dimensional illumination with orthogonal camera-based detection to achieve high-resolution, optically sectioned imaging throughout the sample, with minimal photodamage and at speeds capable of capturing transient biological phenomena. We used SPIM to visualize all muscles in vivo in the transgenic Medaka line Arnie, which expresses green fluorescent protein in muscle tissue. We also demonstrate that SPIM can be applied to visualize the embryogenesis of the relatively opaque
Drosophila melanogaster
in vivo.
Mitochondrial Fusion Intermediates Revealed in Vitro
Authors: Meeusen Shelly , McCaffery J. Michael , Nunnari Jodi
Published: August 2004

The events that occur during the fusion of double-membraned mitochondria are unknown. As an essential step toward determining the mechanism of mitochondrial fusion, we have captured this event in vitro. Mitochondrial outer and inner membrane fusion events were separable and mechanistically distinct, but both required guanosine 5′-triphosphate hydrolysis. Homotypic trans interactions of the ancient outer transmembrane guanosine triphosphatase, Fzo1, were required to promote the fusion of mitochondrial outer membranes, whereas electrical potential was also required for fusion of inner membranes. Our conclusions provide fundamental insights into the molecular events driving mitochondrial fusion and advance our understanding of the evolution of mitochondrial fusion in eukaryotic cells.
Small Interfering RNA-Induced Transcriptional Gene Silencing in Human Cells
Authors: Morris Kevin V. , Chan Simon W.-L. , Jacobsen Steven E. , Looney David J.
Published: August 2004

Small interfering RNA (siRNA) and microRNA silence genes at the transcriptional, posttranscriptional, and/or translational level. Using human tissue culture cells, we show that promoter-directed siRNA inhibits transcription of an integrated, proviral, elongation factor 1alpha (EF1A) promoter–green fluorescent protein reporter gene and of endogenous EF1A. Silencing was associated with DNA methylation of the targeted sequence, and it required either active transport of siRNA into the nucleus or permeabilization of the nuclear envelope by lentiviral transduction. These results demonstrate that siRNA-directed transcriptional silencing is conserved in mammals, providing a means to inhibit mammalian gene function.
Immobilized Cobalt Affinity Chromatography Provides a Novel, Efficient Method for Herpes Simplex Virus Type 1 Gene Vector Purification
Authors: Jiang Canping , Wechuck James B. , Goins William F. , Krisky David M. , Wolfe Darren , Ataai Mohammad M. , Glorioso Joseph C.
Published: August 2004

Herpes simplex virus type 1 (HSV-1) is a promising vector for gene therapy applications, particularly at peripheral nerves, the natural site of virus latency. Many gene vectors require large particle numbers for even early-phase clinical trials, emphasizing the need for high-yield, scalable manufacturing processes that result in virus preparations that are nearly free of cellular DNA and protein contaminants. HSV-1 is an enveloped virus that requires the development of gentle purification methods. Ideally, such methods should avoid centrifugation and may employ selective purification processes that rely on the recognition of a unique envelope surface chemistry. Here we describe a novel method that fulfills these criteria. An immobilized metal affinity chromatography (IMAC) method was developed for the selective purification of vectors engineered to display a high-affinity binding peptide. Feasibility studies involving various transition metal ions (Cu
2+
, Zn
2+
, Ni
2+
, and Co
2+
) showed that cobalt had the most desirable features, which include a low level of interaction with either the normal virus envelope or contaminating DNA and proteins. The introduction of a cobalt-specific recognition element into the virus envelope may provide a suitable target for cobalt-dependent purification. To test this possibility, we engineered a peptide with affinity for immobilized cobalt in frame in the heparan sulfate binding domain of HSV-1 glycoprotein B, which is known to be exposed on the surface of the virion particle and recombined into the viral genome. By optimizing the IMAC loading conditions and reducing cobalt ion leakage, we recovered 78% of the tagged HSV-1 recombinant virus, with a >96% reduction in contaminating proteins and DNA.
Retroviral Vectors Pseudotyped with Severe Acute Respiratory Syndrome Coronavirus S Protein
Authors: Giroglou Tsanan , Cinatl Jindrich , Rabenau Holger , Drosten Christian , Schwalbe Harald , Doerr Hans Wilhelm , von Laer Dorothee
Published: August 2004

The worldwide outbreak of severe acute respiratory syndrome (SARS) was shown to be associated with a novel coronavirus (CoV) now called SARS CoV. We report here the generation of SARS CoV S protein-pseudotyped murine leukemia virus (MLV) vector particles. The wild-type S protein pseudotyped MLV vectors, although at a low efficiency. Partial deletion of the cytoplasmic tail of S dramatically increased infectivity of pseudotypes, with titers only two- to threefold lower than those of pseudotypes generated in parallel with the vesicular stomatitis virus G protein. S-pseudotyped MLV particles were used to analyze viral tropism. MLV(SARS) pseudotypes and wild-type SARS CoV displayed similar cell types and tissue and host restrictions, indicating that the expression of a functional receptor is the major restraint in permissiveness to SARS CoV infection. Efficient gene transfer could be detected in Vero and CaCo2 cells, whereas the level of gene marking of 293T, HeLa, and HepG2 cells was only slightly above background levels. A cat cell line and a dog cell line were not susceptible. Interestingly, PK-15, a porcine kidney cell line, and primary porcine kidney cells were also highly permissive for SARS S pseudotypes and wild-type SARS CoV. This finding suggests that swine may be susceptible to SARS infection and may be a source for infection of humans. Taken together, these results indicate that MLV(SARS) pseudotypes are highly valuable for functional studies of viral tropism and entry and, in addition, can be a powerful tool for the development of therapeutic entry inhibitors without posing a biohazard to human beings.
The N-Terminal Region of the Murine Coronavirus Spike Glycoprotein Is Associated with the Extended Host Range of Viruses from Persistently Infected Murine Cells
Authors: Schickli Jeanne H. , Thackray Larissa B. , Sawicki Stanley G. , Holmes Kathryn V.
Published: August 2004

Although murine coronaviruses naturally infect only mice, several virus variants derived from persistently infected murine cell cultures have an extended host range. The mouse hepatitis virus (MHV) variant MHV/BHK can infect hamster, rat, cat, dog, monkey, and human cell lines but not the swine testis (ST) porcine cell line (J. H. Schickli, B. D. Zelus, D. E. Wentworth, S. G. Sawicki, and K. V. Holmes, J. Virol.
71:
9499-9507, 1997). The spike (S) gene of MHV/BHK had 63 point mutations and a 21-bp insert that encoded 56 amino acid substitutions and a 7-amino-acid insert compared to the parental MHV strain A59. Recombinant viruses between MHV-A59 and MHV/BHK were selected in hamster cells. All of the recombinants retained 21 amino acid substitutions and a 7-amino-acid insert found in the N-terminal region of S of MHV/BHK, suggesting that these residues were responsible for the extended host range of MHV/BHK. Flow cytometry showed that MHV-A59 bound only to cells that expressed the murine glycoprotein receptor CEACAM1a. In contrast, MHV/BHK and a recombinant virus, k6c, with the 21 amino acid substitutions and 7-amino-acid insert in S bound to hamster (BHK) and ST cells as well as murine cells. Thus, 21 amino acid substitutions and a 7-amino-acid insert in the N-terminal region of the S glycoprotein of MHV/BHK confer the ability to bind and in some cases infect cells of nonmurine species.
Specific Residues in the Connector Loop of the Human Cytomegalovirus DNA Polymerase Accessory Protein UL44 Are Crucial for Interaction with the UL54 Catalytic Subunit
Authors: Loregian Arianna , Appleton Brent A. , Hogle James M. , Coen Donald M.
Published: August 2004

The human cytomegalovirus DNA polymerase includes an accessory protein, UL44, which has been proposed to act as a processivity factor for the catalytic subunit, UL54. How UL44 interacts with UL54 has not yet been elucidated. The crystal structure of UL44 revealed the presence of a connector loop analogous to that of the processivity subunit of herpes simplex virus DNA polymerase, UL42, which is crucial for interaction with its cognate catalytic subunit, UL30. To investigate the role of the UL44 connector loop, we replaced each of its amino acids (amino acids 129 to 140) with alanine. We then tested the effect of each substitution on the UL44-UL54 interaction by glutathione
S
-transferase pulldown and isothermal titration calorimetry assays, on the stimulation of UL54-mediated long-chain DNA synthesis by UL44, and on the binding of UL44 to DNA-cellulose columns. Substitutions that affected residues 133 to 136 of the connector loop measurably impaired the UL44-UL54 interaction without altering the ability of UL44 to bind DNA. One substitution, I135A, completely disrupted the binding of UL44 to UL54 and inhibited the ability of UL44 to stimulate long-chain DNA synthesis by UL54. Thus, similar to the herpes simplex virus UL30-UL42 interaction, a residue of the connector loop of the accessory subunit is crucial for UL54-UL44 interaction. However, while alteration of a polar residue of the UL42 connector loop only partially reduced binding to UL30, substitution of a hydrophobic residue of UL44 completely disrupted the UL54-UL44 interaction. This information may aid the discovery of small-molecule inhibitors of the UL44-UL54 interaction.
Factors That Increase the Effective Concentration of CXCR4 Dictate Feline Immunodeficiency Virus Tropism and Kinetics of Replication
Authors: de Parseval Aymeric , Ngo Stacie , Sun Peiqing , Elder John H.
Published: August 2004

The surface glycoprotein (gp95) of the feline immunodeficiency virus (FIV) binds in a strain-specific manner to several cell surface molecules, including CXCR4, heparan sulfate proteoglycans (HSPGs), DC-SIGN, and a 43-kDa cell surface receptor on T cells recently identified as CD134 by M. Shimojima et al. (Science 303:1192-1195, 2004). CXCR4 is the entry receptor in all known cases, and the other molecules act as binding receptors to help facilitate infection. In this report, we confirm and extend the findings regarding CD134 as a primary receptor for FIV. In addition, we show that temperature critically influences the binding properties of FIV gp95 to CXCR4 and HSPGs. The data show that gp95 of the field strain FIV-PPR bound to CXCR4 at 22°C, whereas binding was not detected at 4°C. In contrast, binding of the laboratory adapted FIV-34TF10 gp95 was observed at either 4°C or 22°C, albeit at increased levels at the higher temperature. The level of CXCR4 increased after the temperature was switched from 4 to 22°C, whereas the level of HSPGs decreased, resulting in higher binding of gp95 from both strains to CXCR4 and lower binding of gp95 of FIV-34TF10 to HSPGs (FIV-PPR gp95 does not bind to these molecules). The findings also show that HSPGs facilitate the CXCR4-mediated infectivity of CrFK and G355-5 cells by FIV-34TF10. These two nonlymphoid cell lines express very low levels of CXCR4 and are permissive to FIV-34TF10 but not to productive infection by FIV-PPR. However, overexpression of human CXCR4 in CrFK or G-355-5 cells resulted in extensive cell fusion and infection by FIV-PPR. Taken together, these findings indicate that factors that increase the effective concentration of CXCR4 enhance FIV infectivity and may involve (i) temperature or ligand-induced conformational changes in CXCR4 that enhance SU binding, (ii) coreceptor interactions with gp95 that either alter gp95 conformation to enhance CXCR4 binding and/or raise the localized concentration of receptor or ligand, or (iii) direct increase in CXCR4 concentration via overexpression.
Human Cytomegalovirus UL84 Oligomerization and Heterodimerization Domains Act as Transdominant Inhibitors of
ori
Lyt-Dependent DNA Replication: Evidence that IE2-UL84 and UL84-UL84 Interactions Are Required for Lytic DNA Replication
Authors: Colletti Kelly S. , Xu Yiyang , Cei Sylvia A. , Tarrant Margaret , Pari Gregory S.
Published: August 2004

Human cytomegalovirus (HCMV) UL84 encodes a 75-kDa protein required for
ori
Lyt-dependent DNA replication and interacts with IE2 in infected and transfected cells. UL84 localizes to the nucleus of transfected and infected cells and is found in viral replication compartments. In transient assays it was shown that UL84 can interfere with the IE2-mediated transactivation of the UL112/113 promoter of HCMV. To determine whether UL84 protein-protein interactions are necessary for lytic DNA synthesis, we purified UL84 and used this protein to generate a monoclonal antibody. Using this antibody, we now show that UL84 forms a stable interaction with itself in vivo. The point of self-interaction maps to a region of the protein between amino acids 151 and 200, a domain that contains a series of highly charged amino acid residues. Coimmunoprecipitation assays determined that UL84 interacts with a protein domain present within the first 215 amino acids of IE2. We also show that an intact leucine zipper domain of UL84 is required for a stable interaction with IE2 and UL84 leucine zipper mutants fail to complement
ori
Lyt-dependent DNA replication. UL84 leucine zipper mutants no longer interfere with IE2-mediated transactivation of the UL112/113 promoter, confirming that the leucine zipper is essential for a functional interaction with IE2. In addition, we demonstrate that both the leucine zipper and oligomerization domains of UL84 can act as transdominant-negative inhibitors of lytic replication in the transient assay, strongly suggesting that both an IE2-UL84 and a UL84-UL84 interaction are required for DNA synthesis.
Identification and Characterization of a New Cross-Reactive Human Immunodeficiency Virus Type 1-Neutralizing Human Monoclonal Antibody
Authors: Zhang Mei-Yun , Xiao Xiaodong , Sidorov Igor A. , Choudhry Vidita , Cham Fatim , Zhang Peng Fei , Bouma Peter , Zwick Michael , Choudhary Anil , Montefiori David C. , Broder Christopher C. , Burton Dennis R. , Quinnan Gerald V. , Dimitrov Dimiter S.
Published: August 2004

The identification and characterization of new human monoclonal antibodies (hMAbs) able to neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates from different subtypes may help in our understanding of the mechanisms of virus entry and neutralization and in the development of entry inhibitors and vaccines. For enhanced selection of broadly cross-reactive antibodies, soluble HIV-1 envelope glycoproteins (Envs proteins) from two isolates complexed with two-domain soluble CD4 (sCD4) were alternated during panning of a phage-displayed human antibody library; these two Env proteins (89.6 and IIIB gp140s), and one additional Env (JR-FL gp120) alone and complexed with sCD4 were used for screening. An antibody with relatively long HCDR3 (17 residues), designated m14, was identified that bound to all antigens and neutralized heterologous HIV-1 isolates in multiple assay formats. Fab m14 potently neutralized selected well-characterized subtype B isolates, including JRCSF, 89.6, IIIB, and Yu2. Immunoglobulin G1 (IgG1) m14 was more potent than Fab m14 and neutralized 7 of 10 other clade B isolates; notably, although the potency was on average significantly lower than that of IgG1 b12, IgG1 m14 neutralized two of the isolates with significantly lower 50% inhibitory concentrations than did IgG1 b12. IgG1 m14 neutralized four of four selected clade C isolates with potency higher than that of IgG1 b12. It also neutralized 7 of 17 clade C isolates from southern Africa that were difficult to neutralize with other hMAbs and sCD4. IgG1 m14 neutralized four of seven primary HIV-1 isolates from other clades (A, D, E, and F) much more efficiently than did IgG1 b12; for the other three isolates, IgG b12 was much more potent. Fab m14 bound with high (nanomolar range) affinity to gp120 and gp140 from various isolates; its binding was reduced by soluble CD4 and antibodies recognizing the CD4 binding site (CD4bs) on gp120, and its footprint as defined by alanine-scanning mutagenesis overlaps that of b12. These results suggest that m14 is a novel CD4bs cross-reactive HIV-1-neutralizing antibody that exhibits a different inhibitory profile compared to the only known potent broadly neutralizing CD4bs human antibody, b12, and may have implications for our understanding of the mechanisms of immune evasion and for the development of inhibitors and vaccines.
Regulation of Poliovirus 3C Protease by the 2C Polypeptide
Authors: Banerjee Rajeev , Weidman Mary K. , Echeverri Angela , Kundu Pallob , Dasgupta Asim
Published: August 2004

Poliovirus-encoded nonstructural polypeptide 2C is a multifunctional protein that plays an important role in viral RNA replication. 2C interacts with both intracellular membranes and virus-specific RNAs and has ATPase and GTPase activities. Extensive computer analysis of the 2C sequence revealed that in addition to the known ATPase-, GTPase-, membrane-, and RNA-binding domains it also contains several “serpin” (serine protease inhibitor) motifs. We provide experimental evidence suggesting that 2C is indeed capable of regulating virus-encoded proteases. The purified 2C protein inhibits 3C
pro
-catalyzed cleavage of cellular transcription factors at Q-G sites in vitro. It also inhibits cleavage of a viral precursor by the other viral protease, 2A
pro
. However, at least three cellular proteases appear not to be inhibited by 2C in vitro. The 2C-associated protease inhibitory activity can be depleted by anti-2C antibody. A physical interaction between 2C and His-tagged 3C
pro
can be demonstrated in vitro by coimmunoprecipitation of 2C with anti-His antibody. Deletion analysis suggests that the 2C central and C-terminal domains that include several serpin motifs are important for 3C
pro
-inhibitory activity. To examine the 2C protease inhibitory activity in vivo, stable HeLa cell lines were made that express 2C in an inducible fashion. Infection of 2C-expressing cells with poliovirus led to incomplete (or inefficient) processing of viral precursor polypeptides compared to control cell lines containing the vector alone. These results suggest that 2C can negatively regulate the viral protease 3C
pro
. The possible role of the 2C protease inhibitory activity in viral RNA replication is discussed.
Glycan-Controlled Epitopes of Prion Protein Include a Major Determinant of Susceptibility to Sheep Scrapie
Authors: Moudjou Mohammed , Treguer Eric , Rezaei Human , Sabuncu Elifsu , Neuendorf Erdi , Groschup Martin H. , Grosclaude Jeanne , Laude Hubert
Published: August 2004

A key feature of prion encephalopathies is the accumulation of a misfolded form of the host glycoprotein PrP. Cell-free and cell culture studies have shown that the efficiency of conversion of PrP into the disease-associated form is influenced by its amino acid sequence and also by its carbohydrate moiety. Here, we characterize four novel glycoform-dependent monoclonal antibodies raised against prokaryotic recombinant sheep PrP. We demonstrate that these antibodies discriminate the PrP monoglycosylated species, since two of them recognize molecules that have the first Asn glycosylation site occupied (
mono1
) while the other two recognize molecules glycosylated at the second site (
mono2
). Remarkably, the recognition of PrP by the anti-
mono2
antibodies was strongly influenced by the amino acid present at position 171, i.e., either Gln or Arg. This polymorphism is known to be the main determinant of susceptibility and resistance to scrapie in sheep. Altogether, our findings lead us to propose that each glycan chain controls the accessibility of PrP determinants located close upstream from their attachment site. The monoglycoform-assigned and the allotype-restricted antibodies described here, the first to date, should provide further opportunities to investigate the involvement of each glycan chain in PrP conversion in relation to prion strain diversity and the basis of the resistance conferred by the Arg-171 amino acid.
Adaptive Immunity Is the Primary Force Driving Selection of Equine Infectious Anemia Virus Envelope SU Variants during Acute Infection
Authors: Mealey Robert H. , Leib Steven R. , Pownder Sarah L. , McGuire Travis C.
Published: August 2004

Equine infectious anemia virus (EIAV) is a lentivirus that causes persistent infection in horses. The appearance of antigenically distinct viral variants during recurrent viremic episodes is thought to be due to adaptive immune selection pressure. To test this hypothesis, we evaluated envelope SU cloned sequences from five severe combined immunodeficient (SCID) foals infected with EIAV. Within the SU hypervariable V3 region, 8.5% of the clones had amino acid changes, and 6.4% had amino acid changes within the known cytotoxic T lymphocyte (CTL) epitope Env-RW12. Of all the SU clones, only 3.1% had amino acid changes affecting potential N-linked glycosylation sites. In contrast, a much higher degree of variation was evident in SU sequences obtained from four EIAV-infected immunocompetent foals. Within V3, 68.8% of the clones contained amino acid changes, and 50% of the clones had amino acid changes within the Env-RW12 CTL epitope. Notably, 31.9% of the clones had amino acid changes affecting one or more glycosylation sites. Marked amino acid variation occurred in cloned SU sequences from an immune-reconstituted EIAV-infected SCID foal. Of these clones, 100% had amino acid changes within V3, 100% had amino acid changes within Env-RW12, and 97.5% had amino acid changes affecting glycosylation sites. Analysis of synonymous and nonsynonymous nucleotide substitutions revealed statistically significant differences between SCID and immunocompetent foals and between SCID foals and the reconstituted SCID foal. Interestingly, amino acid selection at one site occurred independently of adaptive immune status. Not only do these data indicate that adaptive immunity primarily drives the selection of EIAV SU variants, but also they demonstrate that other selective forces exist during acute infection.
Phylogenetic Analysis of Polyomavirus Simian Virus 40 from Monkeys and Humans Reveals Genetic Variation
Authors: Forsman Zac H. , Lednicky John A. , Fox George E. , Willson Richard C. , White Zoe S. , Halvorson Steven J. , Wong Connie , Lewis Andrew M. , Butel Janet S.
Published: August 2004

A phylogenetic analysis of 14 complete simian virus 40 (SV40) genomes was conducted in order to determine strain relatedness and the extent of genetic variation. This analysis included infectious isolates recovered between 1960 and 1999 from primary cultures of monkey kidney cells, from contaminated poliovaccines and an adenovirus seed stock, from human malignancies, and from transformed human cells. Maximum-parsimony and distance methods revealed distinct SV40 clades. However, no clear patterns of association between genotype and viral source were apparent. One clade (clade A) is derived from strain 776, the reference strain of SV40. Clade B contains isolates from poliovaccines (strains 777 and Baylor), from monkeys (strains N128, Rh911, and K661), and from human tumors (strains SVCPC and SVMEN). Thus, adaptation is not essential for SV40 survival in humans. The C terminus of the T-antigen (T-ag-C) gene contains the highest proportion of variable sites in the SV40 genome. An analysis based on just the T-ag-C region was highly congruent with the whole-genome analysis; hence, sequencing of just this one region is useful in strain identification. Analysis of an additional 16 strains for which only the T-ag-C gene was sequenced indicated that further SV40 genetic diversity is likely, resulting in a provisional clade (clade C) that currently contains strains associated with human tumors and human strain PML-1. Four other polymorphic regions in the genome were also identified. If these regions were analyzed in conjunction with the T-ag-C region, most of the phylogenetic signal could be captured without complete genome sequencing. This report represents the first whole-genome approach to establishing phylogenetic relatedness among different strains of SV40. It will be important in the future to develop a more complete catalog of SV40 variation in its natural monkey host, to determine if SV40 strains from different clades vary in biological or pathogenic properties, and to identify which SV40 strains are transmissible among humans.
Biophysical and Mutational Analysis of the Putative bZIP Domain of Epstein-Barr Virus EBNA 3C
Authors: West Michelle J. , Webb Helen M. , Sinclair Alison J. , Woolfson Derek N.
Published: August 2004

Epstein-Barr virus nuclear antigen 3C (EBNA 3C) is essential for B-cell immortalization and functions as a regulator of viral and cellular transcription. EBNA 3C contains glutamine-rich and proline-rich domains and a region in the N terminus consisting of a stretch of basic residues followed by a run of leucine residues spaced seven amino acids apart. This N-terminal domain is widely believed to represent a leucine zipper dimerization motif (bZIP). We have performed the first structural and functional analysis of this motif and demonstrated that this domain is not capable of forming stable homodimers. Peptides encompassing the EBNA 3C zipper domain are approximately 54 to 67% α-helical in solution but cannot form dimers at physiologically relevant concentrations. Moreover, the EBNA 3C leucine zipper cannot functionally substitute for another homodimerizing zipper domain in domain-swapping experiments. Our data indicate, however, that the EBNA 3C zipper domain behaves as an atypical bZIP domain and is capable of self-associating to form higher-order α-helical oligomers. Using directed mutagenesis, we also identified a new role for the bZIP domain in maintaining the interaction between EBNA 3C and RBP-Jκ in vivo. Disruption of the helical nature of the zipper domain by the introduction of proline residues reduces the ability of EBNA 3C to inhibit EBNA 2 activation and interact with RBP-Jκ in vivo by 50%, and perturbation of the charge on the basic region completely abolishes this function of EBNA 3C.
Kinetic Analysis of the Interactions between Vaccinia Virus Complement Control Protein and Human Complement Proteins C3b and C4b
Authors: Bernet John , Mullick Jayati , Panse Yogesh , Parab Pradeep B. , Sahu Arvind
Published: August 2004

The vaccinia virus complement control protein (VCP) is an immune evasion protein of vaccinia virus. Previously, VCP has been shown to bind and support inactivation of host complement proteins C3b and C4b and to protect the vaccinia virions from antibody-dependent complement-enhanced neutralization. However, the molecular mechanisms involved in the interaction of VCP with its target proteins C3b and C4b have not yet been elucidated. We have utilized surface plasmon resonance technology to study the interaction of VCP with C3b and C4b. We measured the kinetics of binding of the viral protein to its target proteins and compared it with human complement regulators factor H and sCR1, assessed the influence of immobilization of ligand on the binding kinetics, examined the effect of ionic contacts on these interactions, and sublocalized the binding site on C3b and C4b. Our results indicate that (i) the orientation of the ligand is important for accurate determination of the binding constants, as well as the mechanism of binding; (ii) in contrast to factor H and sCR1, the binding of VCP to C3b and C4b follows a simple 1:1 binding model and does not involve multiple-site interactions as predicted earlier; (iii) VCP has a 4.6-fold higher affinity for C4b than that for C3b, which is also reflected in its factor I cofactor activity; (iv) ionic interactions are important for VCP-C3b and VCP-C4b complex formation; (v) VCP does not bind simultaneously to C3b and C4b; and (vi) the binding site of VCP on C3b and C4b is located in the C3dg and C4c regions, respectively.
Host Cell Gene Expression during Human Immunodeficiency Virus Type 1 Latency and Reactivation and Effects of Targeting Genes That Are Differentially Expressed in Viral Latency
Authors: Krishnan Vyjayanthi , Zeichner Steven L.
Published: August 2004

The existence of reservoirs of cells latently infected with human immunodeficiency virus (HIV) is a major obstacle to the elimination of HIV infection. We studied the changes in cellular gene expression that accompany the reactivation and completion of the lytic viral cycle in cell lines chronically infected with HIV-1. We found that several genes exhibited altered expression in the chronically infected cells compared to the uninfected parental cells prior to induction into lytic replication. A number of gene classes showed increased expression in the chronically infected cells, notably including genes encoding proteasomes, histone deacetylases, and many transcription factors. Following induction of the lytic replication cycle, we observed ordered, time-dependent changes in the cellular gene expression pattern. Approximately 1,740 genes, many of which fall into 385 known pathways, were differentially expressed (P< 0.001), indicating that completion of the HIV replication cycle is associated with distinct, temporally ordered changes in host cell gene expression. Maximum changes were observed in the early and intermediate phases of the lytic replication cycle. Since the changes in gene expression in chronically infected cells suggested that cells latently infected with HIV have a different gene expression profile than corresponding uninfected cells, we studied the expression profiles of three different chronically infected cell lines to determine whether they showed similar changes in common cellular genes and pathways. Thirty-two genes showed significant differential expression in all cell lines studied compared to their uninfected parental cell lines. Notable among them werecdc42andlyn, which were downregulated and are required for HIV Nef binding and viral replication. Other genes previously unrelated to HIV latency or pathogenesis were also differentially expressed. To determine the effects of targeting products of the genes that were differentially expressed in latently infected cells, we treated the latently infected cells with a proteasome inhibitor, clastolactacystin-beta-lactone (CLBL), and an Egr1 activator, resveratrol. We found that treatment with CLBL and resveratrol stimulated lytic viral replication, suggesting that treatment of cells with agents that target cellular genes differentially expressed in latently infected cells can stimulate lytic replication. These findings may offer new insights into the interaction of the latently infected host cell and HIV and suggest therapeutic approaches for inhibiting HIV infection and for manipulating cells latently infected with HIV so as to trigger lytic replication.
Differential Roles of AC2 and AC4 of Cassava Geminiviruses in Mediating Synergism and Suppression of Posttranscriptional Gene Silencing
Authors: Vanitharani Ramachandran , Chellappan Padmanabhan , Pita Justin S. , Fauquet Claude M.
Published: August 2004

Posttranscriptional gene silencing (PTGS) in plants is a natural defense mechanism against virus infection. In mixed infections, virus synergism is proposed to result from suppression of the host defense mechanism by the viruses. Synergistic severe mosaic disease caused by simultaneous infection with isolates of the Cameroon strain of
African cassava mosaic virus
(ACMV-[CM]) and
East African cassava mosaic Cameroon virus
(EACMCV) in cassava and tobacco is characterized by a dramatic increase in symptom severity and a severalfold increase in viral-DNA accumulation by both viruses compared to that in singly infected plants. Here, we report that synergism between ACMV-[CM] and EACMCV is a two-way process, as the presence of the DNA-A component of ACMV-[CM] or EACMCV in
trans
enhanced the accumulation of viral DNA of EACMCV and ACMV-[CM], respectively, in tobacco BY-2 protoplasts. Furthermore, transient expression of ACMV-[CM] AC4 driven by the
Cauliflower mosaic virus
35S promoter (p35S-AC4) enhanced EACMCV DNA accumulation by ∼8-fold in protoplasts, while p35S-AC2 of EACMCV enhanced ACMV-[CM] DNA accumulation, also by ∼8-fold. An
Agrobacterium
-based leaf infiltration assay determined that ACMV-[CM] AC4 and EACMCV AC2, the putative synergistic genes, were able to suppress PTGS induced by green fluorescent protein (GFP) and eliminated the short interfering RNAs associated with PTGS, with a correlated increase in GFP mRNA accumulation. In addition, we have identified AC4 of
Sri Lankan cassava mosaic virus
and AC2 of
Indian cassava mosaic virus
as suppressors of PTGS, indicating that geminiviruses evolved differently in regard to interaction with the host. The specific and different roles played by these AC2 and AC4 proteins of cassava geminiviruses in regulating anti-PTGS activity and their relation to synergism are discussed.
Human Cytomegalovirus Tegument Proteins ppUL82 (pp71) and ppUL35 Interact and Cooperatively Activate the Major Immediate-Early Enhancer
Authors: Schierling Karina , Stamminger Thomas , Mertens Thomas , Winkler Michael
Published: August 2004

The tegument protein ppUL82 (pp71) of human cytomegalovirus (HCMV) has previously been shown to activate the immediate-early transcription of HCMV and to enhance the infectivity of viral DNA. This is concordant with its localization adjacent to promyelocytic leukemia oncogenic domains (PODs) immediately after infection. In a yeast two-hybrid screen, we identified the tegument protein ppUL35 as an interacting partner of ppUL82. The interaction could be confirmed in transfected and infected cells. The domain responsible for interaction was narrowed down to amino acids 447 to 516 within ppUL35, thus allowing both forms of ppUL35 to interact with ppUL82. Immunofluorescence experiments showed a relocalization of ppUL35 from a diffuse nuclear pattern when expressed alone to PODs when expressed together with ppUL82. In accordance with this observation and the role of ppUL82 as a transactivator, we observed a cooperative activation of the HCMV major immediate-early enhancer but not of heterologous viral enhancer elements. These results suggest an important role for this interaction in the stimulation of viral immediate-early gene expression and viral infection.
Pathogenesis of Acute Viral Disease Induced in Fish by Carp Interstitial Nephritis and Gill Necrosis Virus
Authors: Pikarsky Eli , Ronen Ariel , Abramowitz Julia , Levavi-Sivan Berta , Hutoran Marina , Shapira Yechiam , Steinitz Michael , Perelberg Ayana , Soffer Dov , Kotler Moshe
Published: August 2004

A lethal disease of koi and common carp (species
Cyprinus carpio
) has afflicted many fish farms worldwide since 1998, causing severe financial losses. Morbidity and mortality are restricted to common carp and koi and appear in spring and autumn, when water temperatures are 18 to 28°C. We have isolated the virus causing the disease from sick fish, propagated it in koi fin cell culture, and shown that virus from a single clone causes lethal disease in carp and koi upon infection. Intraperitoneal virus injection or bathing the fish in virus-containing water kills 85 to 100% of the fish within 7 to 21 days. This virus is similar to the previously reported koi herpesvirus; however, it has characteristics inconsistent with the herpesvirus family, and thus we have called it carp interstitial nephritis and gill necrosis virus. We examined the pathobiology of this disease in carp by using immunohistochemistry and PCR. We found large amounts of the virus in the kidneys of sick fish and smaller amounts in liver and brain. A rapid increase in the viral load in the kidneys was detected by using both immunofluorescence and semiquantitative PCR. Histological analyses of fish at various times after infection revealed signs of interstitial nephritis as early as 2 days postinfection, which increased in severity up to 10 days postinfection. There was severe gill disease evidenced by loss of villi with accompanying inflammation in the gill rakers. Minimal focal inflammation was noted in livers and brains. This report describes the etiology and pathology of a recently described viral agent in fish.
Model Suggesting that Replication of Influenza Virus Is Regulated by Stabilization of Replicative Intermediates
Authors: Vreede Frank T. , Jung Tanis E. , Brownlee George G.
Published: August 2004

The RNA-dependent RNA polymerase of influenza A virus is responsible for both transcription and replication of negative-sense viral RNA. It is thought that a “switching” mechanism regulates the transition between these activities. We demonstrate that, in the presence of preexisting viral RNA polymerase and nucleoprotein (NP), influenza A virus synthesizes both mRNA (transcription) and cRNA (replication) early in infection. We suggest that there may be no switch regulating the initiation of RNA synthesis and present a model suggesting that nascent cRNA is degraded by host cell nucleases unless it is stabilized by newly synthesized viral RNA polymerase and NP.
The importance of the past in public health
Authors: Scally Gabriel , Womack Justine
Published: August 2004

Study objective:To explore the role of history in public health and its relevance to current practice and professional development.

Design:An analysis of the issues surrounding the poor attention paid to the history of public health by its current practitioners.

Setting:The paper is written from the perspective of practitioners in the UK but has wide applicability.

Main results:The paper makes the case that the current neglect of public health history is to the detriment of public health practice.

Conclusions:There is a strong case for more attention to be paid to public health history in professional formation, development, and communication.
IgA multiple myeloma presenting as non-obstructive jaundice
Authors: Arebi N , Patel B , Aqel N M , Pitcher M C L
Published: August 2004

Multiple myeloma can occasionally present with jaundice. The underlying process may be pancreatic head myeloma infiltration causing obstructive jaundice or hepatic amyloid deposition resulting in cholestatic jaundice. A rare case of myeloma presenting as jaundice due to hepatic myeloma infiltration is reported.
Value of routine duodenal biopsy in diagnosing coeliac disease in patients with iron deficiency anaemia
Authors: Mandal A K , Mehdi I , Munshi S K , Lo T C N
Published: August 2004

Background: Iron deficiency anaemia (IDA) is a recognised feature of coeliac disease in adults and can be its only presentation.

Objective: To determine the yield of routine distal duodenal biopsies in diagnosing coeliac disease in adult and elderly patients with IDA whose endoscopy revealed no upper gastrointestinal cause of iron deficiency.

Study design: Prospective study in a teaching hospital endoscopy unit.

Method: Altogether 504 consecutive patients with IDA, aged 16–80 years, attending for endoscopy were included in this study. At least two distal duodenal biopsies were taken if endoscopy revealed no cause of iron deficiency.

Result: In nine (1.8%) patients duodenal biopsies revealed typical histological features of coeliac disease. Of these, five patients were above 65 years old.

Conclusion: In adult and elderly patients undergoing endoscopy for IDA, the endoscopist should take distal duodenal biopsies to exclude coeliac disease if no upper gastrointestinal cause of anaemia is found. Coeliac disease is not an uncommon cause of IDA in patients >65 years of age and a history of chronic diarrhoea increases diagnostic yield in this age group.
Introduction to history taking and principles of sexual health
Authors: Jones R , Barton S
Published: August 2004

Sexual health has a profound influence over an individual’s physical and psychological wellbeing. There is an increasing need to develop effective strategies to reduce the incidence of sexually transmitted infections (STIs) globally. Many STIs are asymptomatic and yet, highly infectious. The sequelae of such infections may be catastrophic and hence, an integrated multidisciplinary and preventative approach to the control of STIs is clearly necessary. Sexual history taking holds the key to the practice of sexual health medicine. It provides the basis for treatment, prevention, education, and sexual health promotion.
Iliopsoas abscesses
Authors: Mallick I H , Thoufeeq M H , Rajendran T P
Published: August 2004

Iliopsoas abscess is a relatively uncommon condition that can present with vague clinical features. Its insidious onset and occult characteristics can cause diagnostic delays, resulting in high mortality and morbidity. The epidemiology, aetiology, clinical features, and management of iliopsoas abscess are discussed.
THE pT-SPECTRA OF SOME NON-PION SECONDARIES IN HIGH ENERGYNNTONA/AACOLLISIONS AND THE COMBINATIONAL APPROACH
Authors: DE BHASKAR , BHATTACHARYYA S.
Published: August 2004

In continuation of our perusal of the studies on transverse momentum spectra for the main varieties of secondaries from a consistent and comprehensive phenomenological approach, we propose to take up here — after a successful completion of reporting in detail the results (Ref. 4 in the text) on our analyses of the pT-spectra of pions — the studies specially on production of kaons, protons and antiprotons in several proton-induced and nucleus-involved collisions at high energies. The measured data on inclusive cross-sections of kaons, protons and antiprotons from the various published sources have here been assorted first. Next, data on the pT-spectra of the specific secondaries produced in PP and [Formula: see text] reactions have been scanned and analyzed with the help of Hagedorn's model (HM). Thereafter a connector, named here the combinational approach (CA), has been constructed and used to analyze the data on pT-spectra of some major category of non-pion secondaries produced in nucleus–nucleus (AA/AB) collisions at high energies. And these exercises have, finally, led to the modestly successful interpretations of a wide band of data with the revelation of some insightful physical aspects of high energy interactions. The limitations of the approach have also been precisely pointed out in the end.
FEDOSOV SUPERMANIFOLDS: BASIC PROPERTIES AND THE DIFFERENCE IN EVEN AND ODD CASES
Authors: GEYER B. , LAVROV P. M.
Published: August 2004

We study basic properties of supermanifolds endowed with an even (odd) symplectic structure and a connection respecting this symplectic structure. Such supermanifolds can be considered as generalization of Fedosov manifolds to the supersymmetric case. Choosing an appropriate definition of inverse (second-rank) tensor fields on supermanifolds we consider the symmetry behavior of tensor fields as well as the properties of the symplectic curvature and of the Ricci tensor on even (odd) Fedosov supermanifolds. We show that for odd Fedosov supermanifolds the scalar curvature, in general, is nontrivial while for even Fedosov supermanifolds it necessarily vanishes.
TENSIONLESS STRINGS: PHYSICAL FOCK SPACE AND HIGHER SPIN FIELDS
Authors: SAVVIDY G. K.
Published: August 2004

We study the physical Fock space of the tensionless string theory with perimeter action, exploring its new gauge symmetry algebra. The cancellation of conformal anomaly requires the space–time to be 13-dimensional. All particles are massless and there are no tachyon states in the spectrum. The zero mode conformal operator defines the levels of the physical Fock space. All levels can be classified by the highest Casimir operator W of the little group E(11) for massless particles in 11-dimensions. The ground state is infinitely degenerated and contains massless gauge fields of arbitrary large integer spin, realizing the irreducible representations of E(11) of fixed helicity. The excitation levels realize CSR representations of little group E(11) with an infinite number of helicities. After inspection of the first excitation level, which, as we prove, is a physical null state, we conjecture that all excitation levels are physical null states. In this theory the tensor field of the second rank does not play any distinctive role and therefore one can suggest that in this model there is no gravity.