bluuebunny/crossref_metadata_2025 huggingface dataset:
New Insight into the Role of the PhaP Phasin of
Ralstonia eutropha
in Promoting Synthesis of Polyhydroxybutyrate
Authors: York Gregory M. , Stubbe JoAnne , Sinskey Anthony J.
Published: July 2002

Phasins are proteins that are proposed to play important roles in polyhydroxyalkanoate synthesis and granule formation. Here the phasin PhaP of
Ralstonia eutropha
has been analyzed with regard to its role in the synthesis of polyhydroxybutyrate (PHB). Purified recombinant PhaP, antibodies against PhaP, and an
R. eutropha phaP
deletion strain have been generated for this analysis. Studies with the
phaP
deletion strain show that PhaP must accumulate to high levels in order to play its normal role in PHB synthesis and that the accumulation of PhaP to low levels is functionally equivalent to the absence of PhaP. PhaP positively affects PHB synthesis under growth conditions which promote production of PHB to low, intermediate, or high levels. The levels of PhaP generally parallel levels of PHB in cells. The results are consistent with models whereby PhaP promotes PHB synthesis by regulating the surface/volume ratio of PHB granules or by interacting with polyhydroxyalkanoate synthase and indicate that PhaP plays an important role in PHB synthesis from the early stages in PHB production and across a range of growth conditions.
Identification of
fur
and
fldA
Homologs and a
Pasteurella multocida tbpA
Homolog in
Histophilus ovis
and Effects of Iron Availability on Their Transcription
Authors: Ekins Andrew , Niven Donald F.
Published: July 2002

tbpA
,
fur
, and
fldA
homologs from two strains (9L and 3384Y) of the sheep pathogen
Histophilus ovis
were sequenced. The predicted TbpA proteins of these strains are homologs of the
Pasteurella multocida
TbpA protein and collectively represent the second example of a new subfamily of TonB-dependent receptors.
tbpA
transcripts were readily detected by reverse transcription (RT)-PCR with RNA isolated from strain 9L grown under iron-restricted conditions in the presence or absence of bovine transferrin (Tf). However, with strain 3384Y and depending on the primer pair,
tbpA
transcripts were detected by RT-PCR predominantly when the RNA was from cells grown under iron-restricted conditions in the presence of bovine Tf. In both strains, the
fldA
homolog was found to be immediately upstream of
fur
and, based on RT-PCR, these genes are transcribed as a single unit; the availability of iron and the presence or absence of bovine Tf in the growth medium had no apparent effect on the relative amounts of the
fldA
-
fur
transcripts.
Ability of Laboratories To Detect Emerging Antimicrobial Resistance: Proficiency Testing and Quality Control Results from the World Health Organization's External Quality Assurance System for Antimicrobial Susceptibility Testing
Authors: Tenover Fred C. , Mohammed M. Jasmine , Stelling John , O'Brien Thomas , Williams Rosamund
Published: July 2002

The accuracy of antimicrobial susceptibility data submitted by microbiology laboratories to national and international surveillance systems has been debated for a number of years. To assess the accuracy of data submitted to the World Health Organization by users of the WHONET software, the Centers for Disease Control and Prevention distributed six bacterial isolates representing key antimicrobial-resistance phenotypes to approximately 130 laboratories, all but one of which were outside of the United States, for antimicrobial susceptibility testing as part of the World Health Organization's External Quality Assurance System for Antimicrobial Susceptibility Testing. Each laboratory also was asked to submit 10 consecutive quality control values for several key organism-drug combinations. Most laboratories were able to detect methicillin (oxacillin) resistance in
Staphylococcus aureus
, high-level vancomycin resistance in
Enterococcus faecium
, and resistance to extended-spectrum cephalosporins in
Klebsiella pneumoniae
. Many laboratories, particularly those using disk diffusion tests, had difficulty in recognizing reduced susceptibility to penicillin in an isolate of
Streptococcus pneumoniae
. The most difficult phenotype for laboratories to detect was reduced susceptibility to vancomycin in an isolate of
Staphylococcus epidermidis
. The proficiency testing challenge also included a request for biochemical identification of a gram-negative bacillus, which most laboratories recognized as
Enterobacter cloacae
. Although only a small subset of laboratories have submitted their quality control data, it is clear that many of these laboratories generate disk diffusion results for oxacillin when testing
S. aureus
ATCC 25923 and
S. pneumoniae
ATCC 49619 that are outside of the acceptable quality control range. The narrow quality control range for vancomycin also proved to be a challenge for many of the laboratories submitting data; approximately 27% of results were out of range. Thus, it is important to establish the proficiency of laboratories submitting data to surveillance systems in which the organisms are tested locally, particularly for penicillin resistance in pneumococci and glycopeptide resistance in staphylococci.
Posttranscriptional Inhibition of Class I Major Histocompatibility Complex Presentation on Hepatocytes and Lymphoid Cells in Chronic Woodchuck Hepatitis Virus Infection
Authors: Michalak Tomasz I. , Hodgson Paul D. , Churchill Norma D.
Published: July 2002

Woodchuck hepatitis virus (WHV), similar to human hepatitis B virus, causes acute liver inflammation that can progress to chronic hepatitis and hepatocellular carcinoma. WHV also invades cells of the host lymphatic system, where it persists for life. We report here that acute and chronic hepadnavirus hepatitis is characterized by a profound difference in the expression of class I major histocompatibility complex (MHC) molecules on the surface of infected hepatocytes and, notably, lymphoid cells. While acute WHV infection is accompanied by the enhanced hepatocyte surface presentation of class I MHC antigen and upregulated transcription of the relevant hepatic genes, inhibition of class I antigen display on liver cells is a uniform hallmark of chronic WHV infection. This inhibition in chronic hepatitis occurs despite augmented (as in acute infection) expression of hepatic genes for class I MHC heavy chain, β2-microglobulin, and transporters associated with antigen processing (TAP1 and TAP2). Further, the class I antigen inhibition is not related to the histological severity of hepatocellular injury, the extent of lymphocytic infiltrations, the level of intrahepatic gamma interferon induction, or the hepatic WHV load. Importantly, the antigen expression is also inhibited on organ lymphoid cells of chronically infected hosts. The results obtained in this study demonstrate that the defective presentation of class I MHC molecules on cells supporting persistent WHV replication is due to viral posttranscriptional interference. This event may diminish the susceptibility of infected hepatocytes to virus-specific T-cell-mediated elimination, hinder virus clearance, and deregulate the class I MHC-dependent functions of the host immune system. This multifarious effect could be critical for perpetuation of liver damage and evasion of the antiviral immunological surveillance in chronic infection and therefore could be supportive of hepadnavirus persistence.
The Carboxyl Terminus of v-Abl Protein Can Augment SH2 Domain Function
Authors: Warren David , Heilpern Andrew J. , Berg Kent , Rosenberg Naomi
Published: July 2002

Abelson murine leukemia virus (Ab-MLV) transforms NIH 3T3 and pre-B cells via expression of the v-Abl tyrosine kinase. Although the enzymatic activity of this molecule is absolutely required for transformation, other regions of the protein are also important for this response. Among these are the SH2 domain, involved in phosphotyrosine-dependent protein-protein interactions, and the long carboxyl terminus, which plays an important role in transformation of hematopoietic cells. Important signals are sent from each of these regions, and transformation is most likely orchestrated by the concerted action of these different parts of the protein. To explore this idea, we compared the ability of the v-Src SH2 domain to substitute for that of v-Abl in the full-length P120 v-Abl protein and in P70 v-Abl, a protein that lacks the carboxyl terminus characteristic of Abl family members. Ab-MLV strains expressing P70/S2 failed to transform NIH 3T3 cells and demonstrated a greatly reduced capacity to mediate signaling events associated with the Ras-dependent mitogen-activated protein (MAP) kinase pathway. In contrast, Ab-MLV strains expressing P120/S2 were indistinguishable from P120 with respect to these features. Analyses of additional mutants demonstrated that the last 162 amino acids of the carboxyl terminus were sufficient to restore transformation. These data demonstrate that an SH2 domain with v-Abl substrate specificity is required for NIH 3T3 transformation in the absence of the carboxyl terminus and suggest that cooperativity between the extreme carboxyl terminus and the SH2 domain facilitates the transmission of transforming signals via the MAP kinase pathway.
Inhibition of Human Immunodeficiency Virus Type 1 Replication prior to Reverse Transcription by Influenza Virus Stimulation
Authors: Pinto Ligia A. , Blazevic Vesna , Patterson Bruce K. , Mac Trubey C. , Dolan Matthew J. , Shearer Gene M.
Published: July 2002

It is now recognized that, in addition to drug-mediated therapies against human immunodeficiency virus type 1 (HIV-1), the immune system can exert antiviral effects via CD8+T-cell-generated anti-HIV factors. This study demonstrates that (i) supernatants from peripheral blood mononuclear cells (PBMC) stimulated with influenza A virus inhibit replication of CCR5- and CXCR4-tropic HIV-1 isolates prior to reverse transcription; (ii) the HIV-suppressive supernatants can be generated by CD4- or CD8-depleted PBMC; (iii) this anti-HIV activity is partially due to alpha interferon (IFN-α), but not to IFN-γ, IFN-β, the β-chemokines MIP-1α, MIP-1β, and RANTES, or interleukin-16; (iv) the anti-HIV activity is generated equally well by PBMC cultured with either infectious or UV-inactivated influenza A virus; and (v) the antiviral activity can be generated by influenza A-stimulated PBMC from HIV-infected individuals. These findings represent a novel mechanism for inhibition of HIV-1 replication that differs from the previously described CD8 anti-HIV factors (MIP-1α, MIP-1β, RANTES, and CD8 antiviral factor).
Replicase Complex Genes of Semliki Forest Virus Confer Lethal Neurovirulence
Authors: Tuittila Minna T. , Santagati Maria G. , Röyttä Matias , Määttä Jorma A. , Hinkkanen Ari E.
Published: July 2002

Semliki Forest virus (SFV) is a mosquito-transmitted pathogen of small rodents, and infection of adult mice with SFV4, a neurovirulent strain of SFV, leads to lethal encephalitis in a few days, whereas mice infected with the avirulent A7(74) strain remain asymptomatic. In adult neurons, A7(74) is unable to form virions and hence does not reach a critical threshold of neuronal damage. To elucidate the molecular mechanisms of neurovirulence, we have cloned and sequenced the entire 11,758-nucleotide genome of A7(74) and compared it to the highly neurovirulent SFV4 virus. We found several sequence differences and sought to localize determinants conferring the neuropathogenicity by using a panel of chimeras between SFV4 and a cloned recombinant, rA774. We first localized virulence determinants in the nonstructural region by showing that rA774 structural genes combined with the SFV4 nonstructural genome produced a highly virulent virus, while a reciprocal recombinant was asymptomatic. In addition to several amino acid mutations in the nonstructural region, thensp3gene of rA774 displayed an opal termination codon and an in-frame 21-nucleotide deletion close to thensp4junction. Replacement in rA774 of the entirensp3gene with that of SFV4 reconstituted the virulent phenotype, whereas an arginine at the opal position significantly increased virulence, leading to clinical symptoms in mice. Completion of thensp3deletion in rA774 did not increase virulence. We conclude that the opal codon and amino acid mutations other than the deleted residues are mainly responsible for the attenuation of A7(74) and that the attenuating determinants reside entirely in the nonstructural region.
Activation of Transcription Factors NF-κB and NF-IL-6 by Human Immunodeficiency Virus Type 1 Protein R (Vpr) Induces Interleukin-8 Expression
Authors: Roux Philippe , Alfieri Caroline , Hrimech Mohammed , Cohen Eric A. , Tanner Jerome E.
Published: July 2002

Human immunodeficiency virus (HIV)-positive individuals express elevated levels of interleukin-8 (IL-8), which is believed to be responsible for some of the clinical manifestations occurring during AIDS. We report here that virion-derived HIV type 1 (HIV-1) protein R (Vpr) increased IL-8 expression in primary T cells and macrophages, as well as in the T-cell line Jurkat, the monocytic cell line U937, and the epithelial cell line A549. Vpr appeared to increase IL-8 expression and IL-8 promoter activity by activating transcription factors NF-κB and NF-IL-6. Elevated Vpr was also shown to increase transcription of the NF-κB and NF-IL-6 enhancer-containing viral promoters for HIV, cytomegalovirus, and simian virus 40, as well as increase the expression of IL-6 and IL-10 in primary macrophages and in A549 cells, tumor necrosis factor alpha expression in primary T cells, and IL-6 and gamma interferon expression in U937 cells. These results suggest a new role for Vpr in the pathogenesis of HIV infection, namely, the activation of transcription factors NF-IL-6 and NF-κB.
Functional Differences between Human and Bovine Immunodeficiency Virus Tat Transcription Factors
Authors: Bogerd Hal P. , Wiegand Heather L. , Bieniasz Paul D. , Cullen Bryan R.
Published: July 2002

Transcriptional transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter element by the essential viral Tat protein requires recruitment of positive transcription elongation factor b (P-TEFb) to the viral TAR RNA target. The recruitment of P-TEFb, which has been proposed to be necessary and sufficient for activation of viral gene expression, is mediated by the highly cooperative interaction of Tat and cyclin T1, an essential component of P-TEFb, with the HIV-1 TAR element. Species, such as rodents, that encode cyclin T1 variants that are unable to support TAR binding by the Tat-cyclin T1 heterodimer are also unable to support HIV-1 Tat function. In contrast, we here demonstrate that the bovine immunodeficiency virus (BIV) Tat protein is fully able to bind to BIV TAR both in vivo and in vitro in the absence of any cellular cofactor. Nevertheless, BIV Tat can specifically recruit cyclin T1 to the BIV TAR element, and this recruitment is as essential for BIV Tat function as it is for HIV-1 Tat activity. However, because the cyclin T1 protein does not contribute to TAR binding, BIV Tat is able to function effectively in cells from several species that do not support HIV-1 Tat function. Thus, BIV Tat, while apparently dependent on the same cellular cofactor as the Tat proteins encoded by other lentiviruses, is nevertheless unique in terms of the mechanism used to recruit the BIV Tat-cyclin T1 complex to the viral LTR promoter.
The cHS4 Insulator Increases the Probability of Retroviral Expression at Random Chromosomal Integration Sites
Authors: Rivella Stefano , Callegari John A. , May Chad , Tan Cui Wen , Sadelain Michel
Published: July 2002

Retroviruses are highly susceptible to transcriptional silencing and position effects imparted by chromosomal sequences at their integration site. These phenomena hamper the use of recombinant retroviruses as stable gene delivery vectors. As insulators are able to block promoter-enhancer interactions and reduce position effects in some transgenic animals, we examined the effect of an insulator on the expression and structure of randomly integrated recombinant retroviruses. We used the cHS4 element, an insulator from the chicken β-like globin gene cluster, which has been shown to reduce position effects in transgenic
Drosophila
. A large panel of mouse erythroleukemia cells that bear a single copy of integrated recombinant retroviruses was generated without using drug selection. We show that the cHS4 increases the probability that integrated proviruses will express and dramatically decreases the level of de novo methylation of the 5′ long terminal repeat. These findings support a primary role of methylation in the silencing of retroviruses and suggest that cHS4 could be useful in gene therapy applications to overcome silencing of retroviral vectors.
Cell-Specific Modulation of Papovavirus Replication by Tumor Suppressor Protein p53
Authors: Lepik Dina , Ustav Mart
Published: July 2002

Small DNA tumor viruses like human papillomaviruses, simian virus 40, and adenoviruses modulate the activity of cellular tumor suppressor proteins p53 and/or pRB. These viruses replicate as nuclear multicopy extrachromosomal elements during the S phase of the cell cycle, and it has been suggested that inactivation of p53 and pRb is necessary for directing the cells to the S phase. Mouse polyomavirus (Py), however, modulates only the pRB protein activity without any obvious interference with the action of p53. We show here that Py replication was not suppressed by the p53 protein indeed in all tested different mouse cell lines. In addition, E1- and E2-dependent papillomavirus origin replication was insensitive to the action of p53 in mouse cells. We show that in hamster (Chinese hamster ovary) or human (osteosarcoma 143) cell lines the replication of both Py and papillomavirus origins was efficiently blocked by p53. The block of Py replication in human and hamster cells is not caused by the downregulation of large T-antigen expression. The deletion analysis of the p53 protein shows that the RPA binding, proline-rich regulatory, DNA-binding, and oligomerization domains are necessary for p53 action in both replication systems. These results indicate that in mouse cells the p53 protein could be inactive for the suppression of papovavirus replication.
An Adenovirus Inhibitor of Tumor Necrosis Factor Alpha-Induced Apoptosis Complexes with Dynein and a Small GTPase
Authors: Lukashok Sophie A. , Tarassishin Leonid , Li Yongan , Horwitz Marshall S.
Published: July 2002

Adenoviruses (Ad) code for immunoregulatory and cytokine regulatory proteins, one of which is the early region 3, 14.7-kDa protein (Ad E3-14.7K), which has been shown to inhibit tumor necrosis factor alpha-induced apoptosis. In an effort to understand the mechanism of action of Ad E3-14.7K, we previously searched for cell proteins with which it interacted. Three Ad E3-14.7K-interacting proteins (FIP-1, -2, and -3) were isolated. FIP-1 is a small GTPase which was used in this report as bait in the yeast two-hybrid system to find other interacting cell targets. The search resulted in the isolation of a protein, which we called GIP-1 (GTPase-interacting protein) that subsequently was shown to be identical to one of the light-chain components of human dynein (TCTEL1). FIP-1 was able to bind both TCTEL1 and Ad E3-14.7K simultaneously and was necessary to form a complex in which the viral protein was associated with a microtubule-binding motor protein. The functional significance of these interactions is discussed with respect to the steps of the Ad life cycle which are microtubule associated.
Modulation of Dengue Virus Infection in Human Cells by Alpha, Beta, and Gamma Interferons
Authors: Diamond Michael S. , Roberts T. Guy , Edgil Dianna , Lu Betty , Ernst Joel , Harris Eva
Published: July 2002

A role for interferon (IFN) in modulating infection by dengue virus (DV) has been suggested by studies in DV-infected patients and IFN receptor-deficient mice. To address how IFN modulates DV type 2 infection, we have assayed IFN-α, -β, and -γ for the ability to enhance or diminish antibody-independent and antibody-dependent cell infection using a competitive, asymmetric reverse transcriptase-mediated PCR (RT-PCR) assay that quantitates positive and negative strands of viral RNA, a flow cytometric assay that measures viral antigen, and a plaque assay that analyzes virion production. Our data suggest that IFN-α and -β protect cells against DV infection in vitro. Treatment of hepatoma cells with IFN-α or -β decreases viral RNA levels greater than 1,000-fold, the percentage of cells infected 90 to 95%, and the amount of infectious virus secreted 150- to 100,000-fold. These results have been reproduced with several cell types and viral strains, including low-passage isolates. In contrast, IFN-γ has a more variable effect depending on the cell type and pathway of infection. Quantitative RT-PCR experiments indicate that IFN inhibits DV infection by preventing the accumulation of negative-strand viral RNA.
Induction of CD4+T-Cell-Independent Immunoglobulin Responses by Inactivated Influenza Virus
Authors: Sha Zhiyi , Compans Richard W.
Published: July 2002

Through cognate interaction between antigen-specific B-cell and CD4+αβ T cells, the CD4+αβ T cells secrete cytokines that initiate immunoglobulin (Ig) class switching from IgM to IgG. In this study, we show that formalin-inactivated influenza PR8 virus induces virus-specific IgM and IgG responses in the absence of CD4+T cells and that all four subclasses of IgG are produced. The immunized CD4-deficient mice were also found to be completely protected against lethal infection with live, pathogenic influenza virus. The ability of CD4+T-cell-deficient mice to generate these IgG responses was not found to be impaired when these mice were depleted of CD8+T cells with an anti-CD8 monoclonal antibody. In contrast, αβ T-cell-deficient mice (TCRβ−/−) were not found to produce significant amounts of IgG upon immunization with formalin-inactivated PR8 virus. These results suggest that CD4−CD8−double-negative αβ T cells are playing a role in regulating Ig class switching in the absence of CD4+T cells.
Epstein-Barr Virus Nuclear Antigen 3C Activates the Latent Membrane Protein 1 Promoter in the Presence of Epstein-Barr Virus Nuclear Antigen 2 through Sequences Encompassing an Spi-1/Spi-B Binding Site
Authors: Zhao Bo , Sample Clare E.
Published: July 2002

The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) protein is a transcriptional regulator of viral and cellular genes that is essential for EBV-mediated immortalization of B lymphocytes in vitro. EBNA-3C can inhibit transcription through an association with the cellular DNA-binding protein Jκ, a function shared by EBNA-3A and EBNA-3B. Here, we report a mechanism by which EBNA-3C can activate transcription from the EBV latent membrane protein 1 (LMP-1) promoter in conjunction with EBNA-2. Jκ DNA-binding sites were not required for this activation, and a mutant EBNA-3C protein unable to bind Jκ activated transcription as efficiently as wild-type EBNA-3C, indicating that EBNA-3C can regulate transcription through a mechanism that is independent of Jκ. Furthermore, activation of the LMP-1 promoter is a unique function of EBNA-3C, not shared by EBNA-3A and EBNA-3B. The DNA element through which EBNA-3C activates the LMP-1 promoter includes a Spi-1/Spi-B binding site, previously characterized as an important EBNA-2 response element. Although this element has considerable homology to mouse immunoglobulin light chain promoter sequences to which the mouse homologue of Spi-1 binds with its dimerization partner IRF4, we demonstrate that the IRF4-like binding sites in the LMP-1 promoter do not play a role in EBNA-3C-mediated activation. Both EBNA-2 and EBNA-3C were required for transcription mediated through a 41-bp region of the LMP-1 promoter encompassing the Spi binding site. However, EBNA-3C had no effect on transcription mediated in conjunction with the EBNA-2 activation domain fused to the GAL4 DNA-binding domain, suggesting that it does not function as an adapter between EBNA-2 and the cellular transcriptional machinery. Like EBNA-2, EBNA-3C bound directly to both Spi-1 and Spi-B in vitro. This interaction was mediated by a region of EBNA-3C encompassing a likely basic leucine zipper (bZIP) domain and the ets domain of Spi-1 or Spi-B, reminiscent of interactions between bZIP and ets domains of other transcription factors that result in their targeting to DNA. There are many examples of regulation of the hematopoietic-specific Spi transcription factors through protein-protein interactions, and a similar regulation by EBNA-3C, in conjunction with EBNA-2, is likely to be an important and unique contribution of EBNA-3C to EBV-mediated immortalization.
Molecular Epidemiology of Hepatitis B Virus Variants in Nonhuman Primates
Authors: Grethe Stefanie , Heckel Jens-Ove , Rietschel Wolfram , Hufert Frank T.
Published: July 2002

We characterized hepatitis B virus (HBV) isolates from sera of 21 hepatitis B virus surface antigen-positive apes, members of the families
Pongidae
and
Hylobatidae
(19 gibbon spp., 1 chimpanzee, and 1 gorilla). Sera originate from German, French, Thai, and Vietnamese primate-keeping institutions. To estimate the phylogenetic relationships, we sequenced two genomic regions, one located within the pre-S1/pre-S2 region and one including parts of the polymerase and the X protein open reading frames. By comparison with published human and ape HBV isolates, the sequences could be classified into six genomic groups. Four of these represented new genomic groups of gibbon HBV variants. The gorilla HBV isolate was distantly related to the chimpanzee isolate described previously. To confirm these findings, the complete HBV genome from representatives of each genomic group was sequenced. The HBV isolates from gibbons living in different regions of Thailand and Vietnam could be classified into four different phylogenetically distinct genomic groups. The same genomic groups were found in animals from European zoos. Therefore, the HBV infections of these apes might have been introduced into European primate-keeping facilities by direct import of already infected animals from different regions in Thailand. Taken together, our data suggest that HBV infections are indigenous in the different apes. One event involving transmission between human and nonhuman primates in the Old World of a common ancestor of human HBV genotypes A to E and the ape HBV variants might have occurred.
Adeno-Associated Virus Type 5 (AAV5) but Not AAV2 Binds to the Apical Surfaces of Airway Epithelia and Facilitates Gene Transfer
Authors: Zabner Joseph , Seiler Michael , Walters Robert , Kotin Robert M. , Fulgeras Wendy , Davidson Beverly L. , Chiorini John A.
Published: July 2002

In the genetic disease cystic fibrosis, recombinant adeno-associated virus type 2 (AAV2) is being investigated as a vector to transfer CFTR cDNA to airway epithelia. However, earlier work has shown that the apical surface of human airway epithelia is resistant to infection by AAV2, presumably as a result of a lack of heparan sulfate proteoglycans on the apical surface. This inefficiency can be overcome by increasing the amount of vector or by increasing the incubation time. However, these interventions are not very practical for translation into a therapeutic airway-directed vector. Therefore, we examined the efficiency of other AAV serotypes at infecting human airway epithelia. When applied at low multiplicity of infection to the apical surface of differentiated airway epithelia we found that a recombinant AAV5 bound and mediated gene transfer 50-fold more efficiently than AAV2. Furthermore, in contrast to AAV2, AAV5-mediated gene transfer was not inhibited by soluble heparin. Recombinant AAV5 was also more efficient than AAV2 in transferring β-galactosidase cDNA to murine airway and alveolar epithelia in vivo. These data suggest that AAV5-derived vectors bind and mediate gene transfer to human and murine airway epithelia, and the tropism of AAV5 may be useful to target cells that are not permissive for AAV2.
Human Cytomegalovirus Up-Regulates the Phosphatidylinositol 3-Kinase (PI3-K) Pathway: Inhibition of PI3-K Activity Inhibits Viral Replication and Virus-Induced Signaling
Authors: Johnson Robert A. , Wang Xin , Ma Xiu-Li , Huong Shu-Mei , Huang Eng-Shang
Published: July 2002

Infection of quiescent fibroblasts with human cytomegalovirus (HCMV) was found to cause a rapid activation of cellular phosphatidylinositol 3-kinase (PI3-K). Maximum PI3-K activation occurred from 15 to 30 min postinfection. This activation was transient, and by 2 h postinfection (hpi), PI3-K activity had declined to preinfection levels. However, at 4 hpi, a second tier of PI3-K activation was detected, and PI3-K activity remained elevated relative to that of mock-infected cells for the remainder of infection. The cellular kinases Akt and p70S6K and the transcription factor NF-κB were activated in a PI3-K-dependent manner at similar times following HCMV infection. Analysis using UV-irradiated virus indicated that no viral protein synthesis was necessary for the first phase of PI3-K activation, but viral protein expression was required for the second tier of PI3-K activation. Treatment of infected fibroblasts with LY294002, a potent and specific inhibitor of PI3-K kinase activity, caused a 4-log decrease in viral titers. LY294002 did not inhibit viral entry, but it did decrease viral immediate-early gene expression. In addition, the protein levels of two viral early genes required for DNA replication, UL84 and UL44, were significantly lower in the presence of LY294002. Furthermore, viral DNA replication was strongly inhibited by LY294002 treatment. This inhibition of viral DNA replication could be reversed by adding back the products of PI3-K activity (PI-3,4-P2and PI-3,4,5-P3), demonstrating that the effect of LY294002 on the viral life cycle was specifically due to the inhibition of PI3-K activity. These results are the first to suggest that PI3-K mediates HCMV-induced activation of host cell mitogenic pathways. They also provide strong evidence that PI3-K activation is important for initiation of viral DNA replication and completion of the viral lytic life cycle.
Decay Kinetics of Human Immunodeficiency Virus-Specific CD8+T Cells in Peripheral Blood after Initiation of Highly Active Antiretroviral Therapy
Authors: Casazza Joseph P. , Betts Michael R. , Picker Louis J. , Koup Richard A.
Published: July 2002

We measured the longitudinal responses to 95 HLA class I-restricted human immunodeficiency virus (HIV) epitopes and an immunodominant HLA A2-restricted cytomegalovirus (CMV) epitope in eight treatment-naive HIV-infected individuals, using intracellular cytokine staining. Patients were treated with highly active antiretroviral therapy (HAART) for a median of 78 weeks (range, 34 to 121 weeks). Seven of eight patients maintained an undetectable viral load for the duration of therapy. A rapid decline in HIV-specific CD8+T-cell response was observed at initiation of therapy. After an undetectable viral load was achieved, a slower decrease in HIV-specific CD8+T-cell response was observed that was well described by first-order kinetics. The median half-life for the rate of decay was 38.8 (20.3 to 68.0) weeks when data were expressed as percentage of peripheral CD8+T cells. In most cases, data were similar when expressed as the number of responding CD8+T cells per microliter of blood. In subjects who responded to more than one HIV epitope, rates of decline in response to the different epitopes were similar and varied by a factor of 2.2 or less. Discontinuation of treatment resulted in a rapid increase in HIV-specific CD8+T cells. Responses to CMV increased 1.6- and 2.8-fold within 16 weeks of initiation of HAART in two of three patients with a measurable CMV response. These data suggest that HAART quickly starts to restore CD8+T-cell responses to other chronic viral infections and leads to a slow decrease in HIV-specific CD8+T-cell response in HIV-infected patients. The slow decrease in the rate of CD8+T-cell response and rapid increase in response to recurrent viral replication suggest that the decrease in CD8+T-cell response observed represents a normal memory response to withdrawal of antigen.
Expression of DC-SIGN by Dendritic Cells of Intestinal and Genital Mucosae in Humans and Rhesus Macaques
Authors: Jameson Brian , Baribaud Frédéric , Pöhlmann Stefan , Ghavimi Darlene , Mortari Frank , Doms Robert W. , Iwasaki Akiko
Published: July 2002

To better understand the role of dendritic cells (DCs) in human immunodeficiency virus (HIV) transmission at mucosal surfaces, we examined the expressions of the HIV adhesion molecule, dendritic-cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN), its closely related homologue DC-SIGNR, and HIV coreceptors by distinct DC populations in the intestinal and genital tracts of humans and rhesus macaques. We also developed monoclonal antibodies (MAbs) specific for DC-SIGN or DC-SIGNR. In the Peyer's patches, DC-SIGN expression was detected in the interfollicular regions and in clusters of cells in the subepithelial dome regions. DC-SIGN expression was not found on plasmacytoid DCs. DC-SIGNR expression was restricted to endothelial cells in approximately one-third of the capillaries in the terminal ileum. In the vaginal epithelium, Langerhans' cells did not express DC-SIGN, whereas subepithelial DCs in the lamina propria expressed moderate levels of DC-SIGN. Finally, the rectum contained cells that expressed high levels of DC-SIGN throughout the entire thickness of the mucosa, while solitary lymphoid nodules within the rectum showed very little staining for DC-SIGN. Triple-color analysis of rectal tissue indicated that CCR5
+
CD4
+
DC-SIGN
+
DCs were localized just beneath the luminal epithelium. These findings suggest that DC-SIGN
+
DCs could play a role in the transmission of primate lentiviruses in the ileum and the rectum whereas accessibility to DC-SIGN
+
cells is limited in an intact vaginal mucosa. Finally, we identified a MAb that blocked simian immunodeficiency virus interactions with rhesus macaque DC-SIGN. This and other specific MAbs may be used to assess the relevance of DC-SIGN in virus transmission in vivo.
Novel Strategy for Inhibiting Viral Entry by Use of a Cellular Receptor-Plant Virus Chimera
Authors: Khor Ing Wei , Lin Tianwei , Langedijk Johannes P. M. , Johnson John E. , Manchester Marianne
Published: July 2002

The plant virus cowpea mosaic virus (CPMV) has recently been developed as a biomolecular platform to display heterologous peptide sequences. Such CPMV-peptide chimeras can be easily and inexpensively produced in large quantities from experimentally infected plants. This study utilized the CPMV chimera platform to create an antiviral against measles virus (MV) by displaying a peptide known to inhibit MV infection. This peptide sequence corresponds to a portion of the MV binding site on the human MV receptor CD46. The CPMV-CD46 chimera efficiently inhibited MV infection of HeLa cells in vitro, while wild-type CPMV did not. Furthermore, CPMV-CD46 protected mice from mortality induced by an intracranial challenge with MV. Our results indicate that the inhibitory CD46 peptide expressed on the surface of CPMV retains virus-binding activity and is capable of inhibiting viral entry both in vitro and in vivo. The CD46 peptide presented in the context of CPMV is also up to 100-fold more effective than the soluble CD46 peptide at inhibiting MV infection in vitro. To our knowledge, this study represents the first utilization of a plant virus chimera as an antiviral agent.
Polarographic and Voltammetric Determination of 4,4'-Bis[(4-phenylamino-6-methoxy-1,3,5-triazin-2-yl)amino]stilbene-2,2'-disulfonic Acid
Authors: Barek Jiří , Hrnčíř Roman
Published: July 2002

The polarographic and voltammetric behaviour was examined for the title compound, which is the basic component in many commercial optical whitening agents. Dimethylformamide containing 5 vol.% water was chosen as the solvent and tetraethylammonium bromide as the supporting electrolyte. The optimum conditions were found for determining the substance by tast polarography, differential pulse polarography, linear sweep voltammetry on a hanging mercury drop electrode, and differential pulse voltammetry on a hanging mercury drop electrode. The analyte concentration regions for the four techniques were 0.1 - 0.5, 0.01 - 0.5, 0.01 - 0.5 and 0.01 - 0.1 mmol l-1, respectively. Practical applicability of the methods to the determination of the title compound in technical products was verified.
New Procedure for Linear Sweep Voltammetric Determination of Catalytic Reaction Rate Constant. Oxidation of Some Aliphatic Alcohols with Electrolytically Generated Os(VIII)
Authors: Bustin Dušan , Mesároš Štefan , Tomčík Peter , Rievaj Miroslav
Published: July 2002

Oxidation rate of methanol, ethanol and 2-propanol by Os(VIII) in 1 M KOH was determined by evaluating anodic voltammograms of Os(VI) in the presence of the aliphatic alcohols. The proposed method is based on the evaluation of the change of voltammogram shape by catalytic process and on the assumption of its approximation by fractionally integrated voltammograms. This assumption has been confirmed empirically for k'f /a in the interval of <0.04,1>. The rate constants from this interval could be determined with sufficient accuracy. The method is suitable for evaluation of reaction rates slow and moderately fast catalytic reactions. It was compared with other evaluation methods from the literature.
Synthesis and Crystal Structure of Pergolide Sulfoxide
Authors: Čejka Jan , Horák Zdeněk , Pakhomova Svetlana , Kratochvíl Bohumil , Sedmera Petr , Havlíček Vladimír , Cvak Ladislav , Jegorov Alexandr
Published: July 2002

Pergolide sulfoxide (1), a metabolite of pergolide (2), has been prepared by oxidation of 2 with hydrogen peroxide. Its structure was determined by X-ray diffraction and compared with the structures of related ergot alkaloids. Two molecules of pergolide (S)- and (R)-sulfoxide crystallize with one molecule of water in the orthorhombic space group P212121 with a = 12.216(3) Å, b = 13.733(2) Å, c = 22.186(6) Å, Z = 4, V = 3 722(2) Å3.
The Diatomics-in-Molecules Method as a Means for Predicting Potential-Energy-Surface Topolology.A Case Study for the Reaction of C+ with O2
Authors: Polák Rudolf
Published: July 2002

Energy correlation diagrams constructed by means of a Diatomics-in-molecules model, based on the minimum basis of atomic states, indicate some unexpected features of the potential energy surfaces governing the C+ + O2 reaction. Confirmation of the early down-hill character of doublet surfaces and the presence of potential wells in C2v configurations could rise new aspects of the dynamics and mechanism of the reaction, because it is believed that entrance channel effects are very important in this reaction.
Immunostimulant Activity of Peptides Related to Human β-Casein Fragment Gly-Leu-Phe
Authors: Shukla Sushma , Puri Anju , Sahai Ragini , Kundu Bijoy , Tripathi Lalit M. , Srivastava Vishwa M. L.
Published: July 2002

Twenty novel analogues of β-casein fragment (Gly-Leu-Phe) have been synthesised and evaluated in mouse for their impact on macrophage migration and humoral response to sheep red blood cells. As an outcome of the present investigation, compound 1 has been found to be very promising.
Isoenzymes of GMP Kinase from L1210 Cells: Isolation and Characterization
Authors: Krejčová Romana , Horská Květoslava , Votruba Ivan , Holý Antonín
Published: July 2002

Guanylate kinase from a mouse leukemic L1210 cells has been purified nearly to homogeneity for the first time. It consists of five isoenzymes with pI 5.95, 5.50, 5.08, 4.83 and 4.51, respectively. The native enzyme is a monomer with a relative molecular mass 25 000. The kinetic constants Km, Vmax and kcat/Km of isoenzymes were estimated. The purified GMP kinase is absolutely specific to GMP and/or dGMP as phosphate acceptor but has a broad specificity to nucleoside 5'-triphosphates as phosphate donors.
Anodic Oxidation of Novel Hole-Transporting Materials Derived from Tetraarylbenzidines. Electrochemical and Spectroscopic Characterization
Authors: Fáber René , Mielke G. Felix , Rapta Peter , Staško Andrej , Nuyken Oskar
Published: July 2002

Fluorenylidene-linked triarylamines, potential hole-transporting materials, have been prepared by the palladium-catalyzed Hartwig-Buchwald amination. Their redox and spectral properties were investigated in solution, applying cyclic voltammetry, UV-VIS and EPR spectroscopy, and in situ spectroelectrochemical measurements. N,N,N',N'-Tetraphenylbenzidine (1), N,N'-di(1-naphthyl)-N,N'-diphenylbenzidine (2), and triphenylamine (3) served as model substances in the study of the synthesized complex compounds 4 and 5. In structure 4, two triphenylamine centres are linked with a non-conjugated fluorene bridge; in structure 5, two tetraarylbenzidine skeletons with two nitrogens are linked with a conjugated biphenyl-bridge system. In addition, structure 5 contains a non-conjugated fluorene bridge. The presence of the fluorene moiety in the molecular design has a significant influence on the investigated properties of the new materials. In the anodic oxidation of the tetraarylbenzidine-type compounds (1, 2, and 5), two well-defined reversible oxidation peaks were observed. However, the oxidation of the triphenylamine-type structures (3 and 4) is more complex, due to fast consecutive reactions. The dimer-like structures (4 and 5) are characterized by two independent oxidation centres that are simultaneously oxidized at approximately the same potentials. This was confirmed by quantitative cyclovoltammetric as well as UV-VIS investigations.
Benefits and risks of using local anaesthetic for pain relief to allow early return to play in professional football
Authors: Orchard J W
Published: July 2002

Objective:To investigate the risks and benefits of the use of local anaesthetic in a descriptive case series from three professional football (rugby league and Australian football) teams.

Methods:Cases of local anaesthetic use (both injection and topical routes) and complications over a six year period were recorded. Complications were assessed using clinical presentation and also by recording all cases of surgery, incidences of players missing games or leaving the field through injury, and causes of player retirement.

Results:There were 268 injuries for which local anaesthetic was used to allow early return to play. There were 11 minor and six major complications, although none of these were catastrophic or career ending. About 10% of players taking the field did so with the assistance of local anaesthetic. This rate should be considered in isolation and not seen to reflect standard practice by team doctors.

Conclusions:The use of local anaesthetic in professional football may reduce the rates of players missing matches through injury, but there is the risk of worsening the injury, which should be fully explained to players. A procedure should only be used when both the doctor and player consider that the benefits outweigh the risks.
Shifting ethics: debating the incentive question in organ transplantation
Authors: Joralemon Donald
Published: July 2002

The paper reviews the discussion within transplantation medicine about the organ supply and demand problem. The focus is on the evolution of attitudes toward compensation plans from the early 1980s to the present. A vehement rejection on ethical grounds of anything but uncompensated donation—once the professional norm—has slowly been replaced by an open debate of plans that offer financial rewards to persons willing to have their organs, or the organs of deceased kin, taken for transplantation. The paper asks how this shift has occurred and what it tells us about the dynamics of bioethical debates, both within professional circles and in wider public arenas.
Extending the boundaries of the Declaration of Helsinki: a case study of an unethical experiment in a non-medical setting: Table 1
Authors: Richter Elihu D , Barach Paul , Berman Tamar , Ben-David G , Weinberger Zvi
Published: July 2002

To examine the ethical issues involved in governmental decisions with potential health risks, we review the history of the decision to raise the interurban speed limit in Israel in light of its impact on road death and injury. In 1993, the Israeli Ministry of Transportation initiated an “experiment” to raise the interurban speed limit from 90 to 100 kph. The “experiment” did not include a protocol and did not specify cut-off points for early termination in the case of adverse results. After the raise in the speed limit, the death toll on interurban roads rose as a result of a sudden increase in speeds and case fatality rates. The committee's decision is a case study in unfettered human experimentation and public health risks when the setting is non-medical and lacks a defined ethical framework. The case study states the case for extending Helsinki type safeguards to experimentation in non-medical settings.
Medical research in clinical emergency settings in Europe
Authors: Lötjönen S
Published: July 2002

Clinical emergencies necessitate immediate action to avert the danger to the patient's life or health. Emergency patients might be in greatest need of novel therapies, and even presumed willing to assume some risk, but research into emergency conditions should be conducted under commonly accepted principles that fulfil the scientific, ethical, and legal criteria. Such criteria already exist in the US, but are still under development in Europe.

This article introduces criteria upon which trials in emergency settings may be ethically and legally justified in Europe. Based on both legal texts and professional guidelines, the author has established seven conditions for emergency research, of which informed consent and its substitutes, as well as the conditions of direct benefit requirement and necessity, are considered most problematic and therefore analysed more closely. Other conditions include absence of alternative methods, scientific validity, and approval by an ethics committee.
Public reaction to the portrayal of the tobacco industry in the filmThe Insider
Authors: Dixon Helen G , Hill David J , Borland Ron , Paxton Susan J
Published: July 2002

OBJECTIVES

To assess public perceptions of the tobacco industry and behavioural intentions for tobacco use in response to watching the filmThe Insider.

DESIGN

Self administered pre-film survey conducted immediately before viewing and post-film telephone survey conducted within 1–5 weeks of viewing.

SETTING

Two commercial cinemas in Melbourne, Australia.

SUBJECTS

323 cinema patrons were recruited before screening of target films. 182 watchedThe Insider, 141 watchedErin Brockovich.

INTERVENTIONS

Subjects watched one of two films:The Insiderwhich featured information about unethical conduct by the tobacco industry and negative information about the health effects of smoking, or the “control” filmErin Brockovichwhich had an analogous plot without anti-tobacco content.

MAIN OUTCOME MEASURES

Pre-film questionnaire: assessed movie viewing habits, demographic characteristics, smoking status, attitudes towards the tobacco industry, intentions for smoking. Post-film questionnaire: assessed same attitudes and intentions plus questions on the film viewed and perceptions of smoking prevalence.

RESULTS

266 (82%) subjects completed the post-film survey. Attitudes toward the tobacco industry were unfavourable at baseline. Those who sawThe Insiderheld more negative views of business conduct by the tobacco industry than those who sawErin Brockovich, once pre-existing attitudes to the industry were controlled for.The Insideralso appears to have promoted a short term reduction in intentions to smoke.

CONCLUSIONS

Results of this study suggest that if people were recurrently exposed to anti-tobacco content in movies there is potential for a more substantial and lasting impact on attitudes toward the tobacco industry and smoking.