bluuebunny/crossref_metadata_2025 huggingface dataset:
Variable Surface Protein Vmm ofMycoplasma mycoidessubsp.mycoidesSmall Colony Type
Authors: Persson Anja , Jacobsson Karin , Frykberg Lars , Johansson Karl-Erik , Poumarat François
Published: July 2002

A variable surface protein, Vmm, of the bovine pathogenMycoplasma mycoidessubsp.mycoidessmall colony type (M. mycoidesSC) has been identified and characterized. Vmm was specific for the SC biotype and was expressed by 68 of 69 analyzedM. mycoidesSC strains. The protein was found to undergo reversible phase variation at a frequency of 9 × 10−4to 5 × 10−5per cell per generation. Thevmmgene was present in all of the 69 testedM. mycoidesSC strains and encodes a lipoprotein precursor of 59 amino acids (aa), where the mature protein was predicted to be 36 aa and was anchored to the membrane by only the lipid moiety, as no transmembrane region could be identified. DNA sequencing of thevmmgene region from ON and OFF clones showed that the expression of Vmm was regulated at the transcriptional level by dinucleotide insertions or deletions in a repetitive region of the promoter spacer.Vmm-like genes were also found in four closely related mycoplasmas,Mycoplasma capricolumsubsp.capricolum,M. capricolumsubsp. capripneumoniae,Mycoplasmasp. bovine serogroup 7, andMycoplasma putrefaciens. However, Vmm could not be detected in whole-cell lysates of these species, suggesting that the proteins encoded by thevmm-like genes lack the binding epitope for the monoclonal antibody used in this study or, alternatively, that the Vmm-like proteins were not expressed.
Partitioning of Chromosomal DNA during Establishment of Cellular Asymmetry inBacillus subtilis
Authors: Pogliano Joe , Sharp Marc D. , Pogliano Kit
Published: July 2002

The switch from symmetric to asymmetric cell division is a key feature of development in many organisms, includingBacillus subtilissporulation. Here we demonstrate that, prior to the onset of asymmetric cell division, theB. subtilischromosome is partitioned into two unequally sized domains, with the origin-proximal one-third of the future forespore chromosome condensed near one pole of the cell. Asymmetric chromosome partitioning is independent of polar division, as it occurs in cells depleted of FtsZ but depends on two transcription factors that govern the initiation of sporulation, σHand Spo0A-P. It is also independent of chromosome partitioning proteins Spo0J and Soj, suggesting the existence of a novel mechanism controlling chromosome structure. Thus, our results demonstrate that, during sporulation, two separable events prepareB. subtilisfor asymmetric cell division: the relocation of cell division sites to the cell poles and the asymmetric partitioning of the future forespore chromosome.
Quantification of Human Cytomegalovirus DNA by Real-Time PCR
Authors: Gault Elyanne , Michel Yanne , Dehée Axelle , Belabani Chahrazed , Nicolas Jean-Claude , Garbarg-Chenon Antoine
Published: July 2002

A quantitative real-time PCR assay was developed to measure human cytomegalovirus (HCMV) DNA load in peripheral blood leukocytes (PBLs). The HCMV DNA load in PBLs was normalized by means of the quantification of a cellular gene (albumin). The results of the real-time PCR assay correlated with those of the HCMV pp65-antigenemia assay (
P
< 0.0001).
Simultaneous Detection of
Neisseria meningitidis, Haemophilus influenzae,
and
Streptococcus pneumoniae
in Suspected Cases of Meningitis and Septicemia Using Real-Time PCR
Authors: Corless C. E. , Guiver M. , Borrow R. , Edwards-Jones V. , Fox A. J. , Kaczmarski E. B.
Published: July 2002

A single-tube 5′ nuclease multiplex PCR assay was developed on the ABI 7700 Sequence Detection System (TaqMan) for the detection of
Neisseria meningitidis, Haemophilus influenzae,
and
Streptococcus pneumoniae
from clinical samples of cerebrospinal fluid (CSF), plasma, serum, and whole blood. Capsular transport (
ctrA
)
,
capsulation (
bexA
), and pneumolysin (
ply
) gene targets specific for
N. meningitidis, H. influenzae,
and
S. pneumoniae,
respectively, were selected. Using sequence-specific fluorescent-dye-labeled probes and continuous real-time monitoring, accumulation of amplified product was measured. Sensitivity was assessed using clinical samples (CSF, serum, plasma, and whole blood) from culture-confirmed cases for the three organisms. The respective sensitivities (as percentages) for
N. meningitidis, H. influenzae,
and
S. pneumoniae
were 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates. The
ctrA
primers amplified meningococcal serogroups A, B, C, 29E, W135, X, Y, and Z; the
ply
primers amplified pneumococcal serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 14, 15B, 17F, 18C, 19, 20, 22, 23, 24, 31, and 33; and the
bexA
primers amplified
H. influenzae
types b and c. Coamplification of two target genes without a loss of sensitivity was demonstrated. The multiplex assay was then used to test a large number (
n
= 4,113) of culture-negative samples for the three pathogens. Cases of meningococcal,
H. influenzae,
and pneumococcal disease that had not previously been confirmed by culture were identified with this assay. The
ctrA
primer set used in the multiplex PCR was found to be more sensitive (
P
< 0.0001) than the
ctrA
primers that had been used for meningococcal PCR testing at that time.
Antigenic Heterogeneity of the Hepatitis C Virus NS5A Protein
Authors: Dou Xiao-Guang , Talekar Ganesh , Chang Joy , Dai Xing , Li Lixia , Bonafonte Maria-Teresa , Holloway Brian , Fields Howard A. , Khudyakov Yury E.
Published: July 2002

The effect of sequence variability between different types of hepatitis C virus (HCV) on the antigenic properties of the NS5 protein was studied by using recombinant proteins. A strong antigenic region was identified within the HCV NS5A protein at amino acids 2212 to 2313. Forty-five unique sequences encompassing this region were selected from GenBank and were compared to each other. The results of this analysis showed that the primary structure of this strong antigenic region is highly variable. Percent homology between different genotype sequences varied from 40.4 to 72.5%. Thirteen representative sequences from all six HCV genotypes were selected to design synthetic genes coding for this antigenic region. These genes were assembled by PCR from synthetic oligonucleotides and expressed inEscherichia colias hybrid proteins with glutathioneS-transferase. All 13 fusion proteins were purified from bacterial lysates and used to test a panel of anti-HCV positive sera (n= 91) obtained from patients infected with HCV genotypes 1 through 6. All but two proteins immunoreacted with 62 to 93% of HCV anti-NS5-positive serum samples. Although a variable degree of genotype-specific antigenic reactivity was detected, only one protein demonstrated a noticeable preference to immunoreact with antibodies against the homologous HCV genotype. On the other hand, closely related proteins derived from the same subtype or genotype immunoreacted with significantly different efficiency with HCV antibodies. Thus, sequence variability has a profound effect on the antigenic properties of the NS5A immunodominant regions. This observation should be taken into consideration in the development of diagnostic tests for the efficient detection of anti-HCV activity in serum specimens.
A Cell-Line-Specific Defect in the Intracellular Transport and Release of Assembled Retroviral Capsids
Authors: Parker Scott D. , Hunter Eric
Published: July 2002

Retrovirus assembly involves a complex series of events in which a large number of proteins must be targeted to a point on the plasma membrane where immature viruses bud from the cell. Gag polyproteins of most retroviruses assemble an immature capsid on the cytoplasmic side of the plasma membrane during the budding process (C-type assembly), but a few assemble immature capsids deep in the cytoplasm and are then transported to the plasma membrane (B- or D-type assembly), where they are enveloped. With both assembly phenotypes, Gag polyproteins must be transported to the site of viral budding in either a relatively unassembled form (C type) or a completely assembled form (B and D types). The molecular nature of this transport process and the host cell factors that are involved have remained obscure. During the development of a recombinant baculovirus/insect cell system for the expression of both C-type and D-type Gag polyproteins, we discovered an insect cell line (High Five) with two distinct defects that resulted in the reduced release of virus-like particles. The first of these was a pronounced defect in the transport of D-type but not C-type Gag polyproteins to the plasma membrane. High Five cells expressing wild-type Mason-Pfizer monkey virus (M-PMV) Gag precursors accumulate assembled immature capsids in large cytoplasmic aggregates similar to a transport-defective mutant (MA-A18V). In contrast, a larger fraction of the Gag molecules encoded by the M-PMV C-type morphogenesis mutant (MA-R55W) and those of human immunodeficiency virus were transported to the plasma membrane for assembly and budding of virions. When pulse-labeled Gag precursors from High Five cells were fractionated on velocity gradients, they sedimented more rapidly, indicating that they are sequestered in a higher-molecular-mass complex. Compared to Sf9 insect cells, the High Five cells also demonstrate a defect in the release of C-type virus particles. These findings support the hypothesis that host cell factors are important in the process of Gag transport and in the release of enveloped viral particles.
Three Spliced mRNAs of TT Virus Transcribed from a Plasmid Containing the Entire Genome in COS1 Cells
Authors: Kamahora Toshio , Hino Shigeo , Miyata Hironori
Published: July 2002

A permuted whole-genome construct of a TT virus (TTV), named VT416, had 3,852 nucleotides (nt) 98.2% similar to the prototype TA278 genome. To allow the transcription of TTV from the internal promoter, pBK*VT416(1.3G), carrying 1.3 units of VT416, was constructed. The poly(A)
+
RNAs expressed in COS1 cells 48 h posttransfection contained three TTV mRNA species 3.0, 1.2, and 1.0 kb in length, which were recovered in the 13 DNA clones from a λ phage cDNA library. These mRNAs in the antigenomic orientation possessed in common the 3′ terminus downstream of a poly(A) signal (A
3073
ATAAA) and the 5′ terminus downstream of a cap site (C
98
ACTTC). A common splicing to join nt 185 with nt 277 was detected in all mRNAs. The coding region of the largest open reading frame (ORF) was maintained in 3.0-kb mRNA, because this splicing was located upstream of its initiation codon (A
589
TG). The second splicing was detected in 1.2-kb mRNA to join nt 711 with nt 2374 and in 1.0-kb mRNA to bind nt 711 to nt 2567. They linked a proposed ORF2 to another ORF for creating new ORFs over nt 2374 to 2872 in frame 2 and nt 2567 to 3074 in frame 3. The donor and acceptor sites of all three splicings matched the consensus sequence and were conserved in most of the 16 TTVs of distinct genotypes retrieved from the database. The observed transcription profile is unique to TTV among known members in the family
Circoviridae
.
Epstein-Barr Virus LMP-1 Natural Sequence Variants Differ in Their Potential To Activate Cellular Signaling Pathways
Authors: Fielding Ceri A. , Sandvej Kristian , Mehl Anja , Brennan Paul , Jones Matthew , Rowe Martin
Published: July 2002

The latent membrane protein 1 (LMP-1) oncogene of Epstein-Barr virus (EBV) is believed to contribute to the development of many EBV-associated tumors, and there is evidence that sequence variation can affect some functions of LMP-1. Most studies have been restricted to the prototype B95.8 LMP-1 gene and genes isolated from EBV of nasopharyngeal carcinoma (NPC) patients. Here, we analyzed the signaling functions of LMP-1 from a panel of nine EBV isolates, including representatives of four defined groups of European LMP-1 variants (groups A to D [K. Sandvej, J. W. Gratama, M. Munch, X. G. Zhou, R. L. Bolhuis, B. S. Andresen, N. Gregersen, and S. Hamilton-Dutoit, Blood 90:323–330, 1997]) and Chinese NPC-derived LMP-1. Chinese and group D variants activated the transcription factor NF-κB two- to threefold more efficiently than B95.8 LMP-1, while Chinese, group B, and group D variants similarly activated activator protein 1 (AP-1) transcription more efficiently than did B95.8 LMP-1. However, there were no amino acid substitutions in the core binding regions for tumor necrosis factor receptor-associated adapter proteins known to mediate NF-κB and AP-1 activation. In contrast, despite sequence variation in the proposed Janus kinase 3 binding region, STAT activation was remarkably constant among the panel of LMP-1 variants. Analysis of the induction of CD54 (intercellular adhesion molecule 1) protein expression by the LMP-1 variants showed differences that did not correlate with either NF-κB or AP-1. Therefore, while the defined sequence variant groups do correlate with LMP-1 function, the results highlight the fact that the relationship between sequence variation and signaling function is extremely complex. It appears unlikely that one particular amino acid substitution or deletion will define a disease-associated variant of LMP-1.
Characterization of Retrovirus-Host DNA Junctions in Cells Deficient in Nonhomologous-End Joining
Authors: Taganov Konstantin , Daniel René , Katz Richard A. , Favorova Olga , Skalka Anna Marie
Published: July 2002

Formation of stably integrated proviruses is inefficient in cells that are defective in the cellular nonhomologous end-joining (NHEJ) DNA repair pathway (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644–647, 1999; R. Daniel, R. A. Katz, and A. M. Skalka, Mol. Cell. Biol. 21:1164–1172, 2001). However, the requirement for NHEJ function is not absolute, as 10 to 20% of infected NHEJ-deficient cells can express retrovirus- transduced reporter genes in a stable fashion. To learn more about the compensatory mechanism by which viral DNA may be incorporated into the host cell genome, we analyzed the nucleotide sequences of provirus-host DNA junctions in singly infected NHEJ-deficient cell clones. The results showed that the proviral DNA ends in all NHEJ-deficient clones had the normal 5′TG … CA3′ sequence. In addition, 14 of the 19 proviruses analyzed were flanked by a 6-bp direct repeat of host sequences, as is characteristic for avian sarcoma virus integration. These results indicate that the DNA repair pathway which compensates for loss of NHEJ in these transductants does not introduce any gross abnormalities at the provirus-host DNA junctions.
Effects of Homology Length in the Repeat Region on Minus-Strand DNA Transfer and Retroviral Replication
Authors: Dang Que , Hu Wei-Shau
Published: July 2002

Homology between the two repeat (R) regions in the retroviral genome mediates minus-strand DNA transfer during reverse transcription. We sought to define the effects of R homology lengths on minus-strand DNA transfer. We generated five murine leukemia virus (MLV)-based vectors that contained identical sequences but different lengths of the 3′ R (3, 6, 12, 24 and 69 nucleotides [nt]); 69 nt is the full-length MLV R. After one round of replication, viral titers from the vector with a full-length downstream R were compared with viral titers generated from the other four vectors with reduced R lengths. Viral titers generated from vectors with R lengths reduced to one-third (24 nt) or one-sixth (12 nt) that of the wild type were not significantly affected; however, viral titers generated from vectors with only 3- or 6-nt homology in the R region were significantly lower. Because expression and packaging of the RNA were similar among all the vectors, the differences in the viral titers most likely reflected the impact of the homology lengths on the efficiency of minus-strand DNA transfer. The molecular nature of minus-strand DNA transfer was characterized in 63 proviruses. Precise R-to-R transfer was observed in most proviruses generated from vectors with 12-, 24-, or 69-nt homology in R, whereas aberrant transfers were predominantly used to generate proviruses from vectors with 3- or 6-nt homology. Reverse transcription using RNA transcribed from an upstream promoter, termed read-in RNA transcripts, resulted in most of the aberrant transfers. These data demonstrate that minus-strand DNA transfer is homology driven and a minimum homology length is required for accurate and efficient minus-strand DNA transfer.
Impairment of Gag-Specific CD8+T-Cell Function in Mucosal and Systemic Compartments of Simian Immunodeficiency Virus mac251- and Simian-Human Immunodeficiency Virus KU2-Infected Macaques
Authors: Hel Zdenek , Nacsa Janos , Kelsall Brian , Tsai Wen-Po , Letvin Norman , Parks Robyn Washington , Tryniszewska Elzbieta , Picker Louis , Lewis Mark G. , Edghill-Smith Yvette , Moniuszko Marcin , Pal Ranajit , Stevceva Liljana , Altman John D. , Allen Todd M. , Watkins David , Torres José V. , Berzofsky Jay A. , Belyakov Igor M. , Strober Warren , Franchini Genoveffa
Published: July 2002

The identification of several simian immunodeficiency virus mac251 (SIVmac251) cytotoxic T-lymphocyte epitopes recognized by CD8+T cells of infected rhesus macaques carrying the Mamu-A*01 molecule and the use of peptide-major histocompatibility complex tetrameric complexes enable the study of the frequency, breadth, functionality, and distribution of virus-specific CD8+T cells in the body. To begin to address these issues, we have performed a pilot study to measure the virus-specific CD8+and CD4+T-cell response in the blood, lymph nodes, spleen, and gastrointestinal lymphoid tissues of eight Mamu-A*01-positive macaques, six of those infected with SIVmac251and two infected with the pathogenic simian-human immunodeficiency virus KU2. We focused on the analysis of the response to peptide p11C, C-M (Gag 181), since it was predominant in most tissues of all macaques. Five macaques restricted viral replication effectively, whereas the remaining three failed to control viremia and experienced a progressive loss of CD4+T cells. The frequency of the Gag 181 (p11C, C→M) immunodominant response varied among different tissues of the same animal and in the same tissues from different animals. We found that the functionality of this virus-specific CD8+T-cell population could not be assumed based on the ability to specifically bind to the Gag 181 tetramer, particularly in the mucosal tissues of some of the macaques infected by SIVmac251that were progressing to disease. Overall, the functionality of CD8+tetramer-binding T cells in tissues assessed by either measurement of cytolytic activity or the ability of these cells to produce gamma interferon or tumor necrosis factor alpha was low and was even lower in the mucosal tissue than in blood or spleen of some SIVmac251-infected animals that failed to control viremia. The data obtained in this pilot study lead to the hypothesis that disease progression may be associated with loss of virus-specific CD8+T-cell function.
Effector Function Activities of a Panel of Mutants of a Broadly Neutralizing Antibody against Human Immunodeficiency Virus Type 1
Authors: Hezareh Marjan , Hessell Ann J. , Jensen Richard C. , van de Winkel Jan G. J. , Parren Paul W. H. I.
Published: July 2002

The human antibody immunoglobulin G1 (IgG1) b12 neutralizes a broad range of human immunodeficiency virus-type 1 (HIV-1) isolates in vitro and is able to protect against viral challenge in animal models. Neutralization of free virus, which is an antiviral activity of antibody that generally does not require the antibody Fc fragment, likely plays an important role in the protection observed. The role of Fc-mediated effector functions, which may reduce infection by inducing phagocytosis and lysis of virions and infected cells, however, is less clear. To investigate this role, we constructed a panel of IgG1 b12 mutants with point mutations in the second domain of the antibody heavy chain constant region (CH2). These mutations, as expected, did not affect gp120 binding or HIV-1 neutralization. IgG1 b12 mediated strong antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of HIV-1-infected cells, but these activities were reduced or abrogated for the antibody mutants. Two mutants were of particular interest. K322A showed a twofold reduction in FcγR binding affinity and ADCC, while C1q binding and CDC were abolished. A double mutant (L234A, L235A) did not bind either FcγR or C1q, and both ADCC and CDC functions were abolished. In this study, we confirmed that K322 forms part of the C1q binding site in human IgG1 and plays an important role in the molecular interactions leading to complement activation. Less expectedly, we demonstrate that the lower hinge region in human IgG1 has a strong modulating effect on C1q binding and CDC. The b12 mutants K322A and L234A, L235A are useful tools for dissecting the in vivo roles of ADCC and CDC in the anti-HIV-1 activity of neutralizing antibodies.
Intracellular Trafficking of the UL11 Tegument Protein of Herpes Simplex Virus Type 1
Authors: Loomis Joshua S. , Bowzard J. Bradford , Courtney Richard J. , Wills John W.
Published: July 2002

Growing evidence indicates that herpes simplex virus type 1 (HSV-1) acquires its final envelope in the trans-Golgi network (TGN). During the envelopment process, the viral nucleocapsid as well as the envelope and tegument proteins must arrive at this site in order to be incorporated into assembling virions. To gain a better understanding of how these proteins associate with cellular membranes and target to the correct compartment, we have been studying the intracellular trafficking properties of the small tegument protein encoded by the U
L
11 gene of HSV-1. This 96-amino-acid, myristylated protein accumulates on the cytoplasmic face of internal membranes, where it is thought to play a role in nucleocapsid envelopment and egress. When expressed in the absence of other HSV-1 proteins, the UL11 protein localizes to the Golgi apparatus, and previous deletion analyses have revealed that the membrane-trafficking information is contained within the first 49 amino acids. The goal of this study was to map the functional domains required for proper Golgi membrane localization. In addition to N-terminal myristylation, which allows for weak membrane binding, UL11 appears to be palmitylated on one or more of three consecutive N-terminal cysteines. Using membrane-pelleting experiments and confocal microscopy, we show that palmitylation of UL11 is required for both Golgi targeting specificity and strong membrane binding. Furthermore, we found that a conserved acidic cluster within the first half of UL11 is required for the recycling of this tegument protein from the plasma membrane to the Golgi apparatus. Taken together, our results demonstrate that UL11 has highly dynamic membrane-trafficking properties, which suggests that it may play multiple roles on the plasma membrane as well as on the nuclear and TGN membranes.
Increased Readthrough Transcription across the Simian Virus 5 M-F Gene Junction Leads to Growth Defects and a Global Inhibition of Viral mRNA Synthesis
Authors: Parks Griffith D. , Ward Kimberly R. , Rassa John C.
Published: July 2002

Recombinant simian virus 5 (rSV5) mutants containing substitutions in the M-F intergenic region were generated to determine the effect of increased readthrough transcription on the paramyxovirus growth cycle. We have previously shown, using an SV5 dicistronic minigenome, that replacement of the 22-base M-F intergenic region with a foreign sequence results in a template (Rep22) that directs very high levels of M-F readthrough transcription. An rSV5 containing the Rep22 substitution grew slower and to final titers that were 50- to 80-fold lower than those of wild-type (WT) rSV5. Cells infected with the Rep22 virus produced very low levels of monocistronic M and F mRNA, consistent with the M-F readthrough phenotype. Surprisingly, Rep22 virus-infected cells also displayed a global decrease in the accumulation of viral mRNA from genes located upstream and downstream of the M-F junction, and overall viral protein synthesis was reduced. Second-site revertants of the Rep22 virus that had regained WT transcription and growth properties contained a single base substitution that increased the M gene end U tract from four to eight residues, suggesting that the growth defects originated from higher-than-normal M-F readthrough transcription. Thus, the primary growth defect for the Rep22 virus appears to be in viral RNA synthesis and not in morphogenesis. A second rSV5 virus (G14), which contained a different foreign M-F intergenic sequence, grew to similar or slightly higher titers than WT rSV5 in some cell types and produced ∼1.5- to 2-fold more mRNA and viral protein. The data support the hypothesis that inhibition of Rep22 virus growth is due to increased access by the polymerase to the 5′ end of the genome and to the resulting overexpression of L protein. We propose that the elevated naturally occurring M-F readthrough which is characteristic of many paramyxoviruses serves as a mechanism to fine-tune the level of polymerase that is optimal for virus growth.
Complete Inhibition of Tobamovirus Multiplication by Simultaneous Mutations in Two Homologous Host Genes
Authors: Yamanaka Takuya , Imai Takahiro , Satoh Rena , Kawashima Arata , Takahashi Miki , Tomita Kayo , Kubota Kenji , Meshi Tetsuo , Naito Satoshi , Ishikawa Masayuki
Published: July 2002

The
TOM1
gene of
Arabidopsis thaliana
encodes a putative multipass transmembrane protein which is necessary for the efficient multiplication of tobamoviruses. We have previously shown that mutations severely destructive to the
TOM1
gene reduce tobamovirus multiplication to low levels but do not impair it completely. In this report, we subjected one of the
tom1
mutants (
tom1-1
) to another round of mutagenesis and isolated a new mutant which did not permit a detectable level of tobamovirus multiplication. In addition to
tom1-1
, this mutant carried a mutation referred to as
tom3-1
. Positional cloning showed that
TOM3
was one of two
TOM1
-like genes in
Arabidopsis
. Based on the similarity between the amino acid sequences of TOM1 and TOM3, together with the results of a Sos recruitment assay suggesting that both TOM1 and TOM3 bind tobamovirus-encoded replication proteins, we propose that TOM1 and TOM3 play parallel and essential roles in the replication of tobamoviruses.
Herpes Simplex Virus Tegument Protein US11 Interacts with Conventional Kinesin Heavy Chain
Authors: Diefenbach Russell J. , Miranda-Saksena Monica , Diefenbach Eve , Holland David J. , Boadle Ross A. , Armati Patricia J. , Cunningham Anthony L.
Published: July 2002

Little is known about the mechanisms of transport of neurotropic herpesviruses, such as herpes simplex virus (HSV), varicella-zoster virus, and pseudorabies virus, within neurons. For these viruses, which replicate in the nucleus, anterograde transport from the cell body of dorsal root ganglion (DRG) neurons to the axon terminus occurs over long distances. In the case of HSV, unenveloped nucleocapsids in human DRG neurons cocultured with autologous skin were observed by immunoelectron microscopy to colocalize with conventional ubiquitous kinesin, a microtubule-dependent motor protein, in the cell body and axon during anterograde axonal transport. Subsequently, four candidate kinesin-binding structural HSV proteins were identified (VP5, VP16, VP22, and US11) using oligohistidine-tagged human ubiquitous kinesin heavy chain (uKHC) as bait. Of these viral proteins, a direct interaction between uKHC and US11 was identified. In vitro studies identified residues 867 to 894 as the US11-binding site in uKHC located within the proposed heptad repeat cargo-binding domain of uKHC. In addition, the uKHC-binding site in US11 maps to the C-terminal RNA-binding domain. US11 is consistently cotransported with kinetics similar to those of the capsid protein VP5 into the axons of dissociated rat neurons, unlike the other tegument proteins VP16 and VP22. These observations suggest a major role for the uKHC-US11 interaction in anterograde transport of unenveloped HSV nucleocapsids in axons.
Coxsackievirus B3 Replication Is Reduced by Inhibition of the Extracellular Signal-Regulated Kinase (ERK) Signaling Pathway
Authors: Luo Honglin , Yanagawa Bobby , Zhang Jingchun , Luo Zongshu , Zhang Mary , Esfandiarei Mitra , Carthy Christopher , Wilson Janet E. , Yang Decheng , McManus Bruce M.
Published: July 2002

Coxsackievirus B3 (CVB3) is the most common human pathogen for viral myocarditis. We have previously shown that the signaling protein p21rasGTPase-activating protein (RasGAP) is cleaved and that mitogen-activated protein kinases (MAPKs) ERK1/2 are activated in the late phase of CVB3 infection. However, the role of intracellular signaling pathways in CVB3-mediated myocarditis and the relative advantages of such pathways to host or virus remain largely unclear. In this study we extended our prior studies by examining the interaction between CVB3 replication and intracellular signaling pathways in HeLa cells. We observed that CVB3 infection induced a biphasic activation of ERK1/2, early transient activation versus late sustained activation, which were regulated by different mechanisms. Infection by UV-irradiated, inactivated virus capable of receptor binding and endocytosis triggered early ERK1/2 activation, but was insufficient to trigger late ERK1/2 activation. By using a general caspase inhibitor (zVAD.fmk) we further demonstrated that late ERK1/2 activation was not a result of CVB3-mediated caspase cleavage. Treatment of cells with U0126, a selective inhibitor of MAPK kinase (MEK), significantly inhibited CVB3 progeny release and decreased virus protein production. Furthermore, inhibition of ERK1/2 activation circumvented CVB3-induced apoptosis and viral protease-mediated RasGAP cleavage. Taken together, these data suggest that ERK1/2 activation is important for CVB3 replication and contributes to virus-mediated changes in host cells. Our findings demonstrate coxsackievirus takeover of a particular host signaling mechanism and uncover a prospective approach to stymie virus spread and preserve myocardial integrity.
Water Transport and Sorption in Polyimide-Polysiloxane Copolymers
Authors: Sysel Petr , Hynek Vladimír , Šípek Milan
Published: July 2002

Water transport through and sorption in films of polyimide-polysiloxane block copolymers were studied at 25 °C and atmospheric pressure. The permeability and diffusion coefficients and, to a certain extent, also the sorbed amount were found to depend on the content of the polysiloxane blocks.
Diffusion of 1,2-Dichloroethane in Particles of Activated Carbon
Authors: Bobok Daniel , Besedová Eva
Published: July 2002

Simultaneous diffusion and adsorption of 1,2-dichloroethane from air into activated carbon particles was studied at ambient temperature. The measurements were carried out in adsorbers with a bed height equal to the diameter of activated carbon particles. On the basis of the assumption of diffusion and adsorption in a quasihomogenous porous medium, effective diffusion coefficients were calculated. Their values are linearly dependent on the concentration of adsorptive in the inlet gas. For the evaluation of errors, mass transfer resistances in the fluid and solid phases were calculated. The maximum value of the former did not exceed 12.5% of the overall resistance.
Ab initio Calculation of Transport Properties of Supercritical Carbon Dioxide
Authors: Steinebrunner Gerold , Dyson Anthony J. , Kirchner Barbara , Huber Hanspeter
Published: July 2002

Self diffusion, shear viscosity and thermal conductivity of carbon dioxide are determined fully ab initio using two different intermolecular potential energy surfaces. These properties are calculated using the time-correlation formalism in classical equilibrium molecular dynamics simulations. The self diffusion constant is in addition determined from the Einstein relation. For the shear viscosity we use two different models of momentum localization: at the center of mass of the molecules, or at each atom. For the thermal conductivity we apply the formulae for rigid and flexible molecules assuming energy localization at the center of mass of the molecules. The results obtained are in good agreement with experiment. A fully ab initio calculation of transport properties allows for a prediction of these quantities even at state points where experiments are hardly possible.
Helical Phenanthrenes, Part 5. Synthesis of Pentahelicene-7,8-dione via Intramolecular Benzoin Condensation
Authors: Modler-Spreitzer Anja , Fritsch Rainer , Mannschreck Albrecht
Published: July 2002

The intramolecular benzoin condensation of [1,1'-binaphthyl]-2,2'-dicarbaldehyde (5), followed by oxidation with air, provides a new synthesis of (MP)-pentahelicene-7,8-dione (1). With reference to the original preparation of this quinoneviaacyloin condensation, its present yield (73% for the ring-closing step) is considerably increased.
Plots of the Spin-Lattice Relaxation Time and Nuclear Overhauser Enhancement NMR Values Yielding a Spread Distribution of the Rotational Correlation Time
Authors: Jakeš Jaromír
Published: July 2002

It is shown, that the plots of ω/T1and NOE × ω/T1versus-log ω give the distribution of rotational correlation time τ plottedversuslog τ and spread by (rather wide) "slit" functions the form and shape of which are independent of the position on the log τ axis. When plotting data at various temperatures, temperature shift factorsaTand the plotsversus-log (ωaT) should be used. The plots permit to check the data compatibility and are helpful in studying molecular dynamics. Examples of the plots are given. One example stimulated a consideration of the segmental motion dynamics, which showed that the Hall-Helfand distribution is inappropriate for a description of the orientational relaxation of the non-backbone (C-H) bonds in a polymer chain.
Separation of Hydrophilic Oligomers and Polymers Using Monodisperse Poly(2,3-dihydroxypropyl Methacrylate-co-Ethylene Dimethacrylate) Beads via Normal-Phase and Hydrophilic-Interaction HPLC
Authors: Švec František , Petro Miroslav , Fréchet Jean M. J.
Published: July 2002

Microparticulate, monosized, and macroporous poly(2,3-dihydroxypropyl methacrylate-co-ethylene dimethacrylate) beads have been used as a stationary phase for HPLC separations of hydrophilic oligomers and polymers. Homogeneous coverage of the sorbent surface with a large number of chemically equivalent diol functionalities affords suitable retentivity in both normal-phase and hydrophilic-interaction chromatographic modes and enables the separations of water-soluble oligomers and polymers. Chromatographic properties of this stationary phase are demonstrated on a variety of separations of poly(oxyalkylene)s, polyvinylpyrrolidones, and polysaccharides.
Does early detection of otitis media with effusion prevent delayed language development?
Authors: Butler C C , MacMillan H
Published: July 2002

OBJECTIVE

To consider whether earlier detection of otitis media with effusion (OME) in asymptomatic children in the first 4 years of life prevents delayed language development.

METHODS

MEDLINE and other databases were searched and relevant references from articles reviewed. Critical appraisal and consensus development were in accordance with the methods of the Canadian Task Force on Preventive Health Care.

RESULTS

No randomised controlled trials assessing the overall screening for OME and early intervention to prevent delay in acquiring language were identified, although one trial evaluated treatment in a screened population and found no benefit. The “analytic pathway” approach was therefore used, where evidence is evaluated for individual steps in a screening process. The evidence supporting the use of tools for early detection such as tympanometry, microtympanometry, acoustic reflectometry, and pneumatic otoscopy in the first 4 years of life is unclear. Some treatments (mucolytics, antibiotics, steroids) resulted in the short term resolution of effusions as measured by tympanometry. Ventilation tubes resolved effusions and improved hearing. Ventilation tubes in children with hearing loss associated with OME benefited children in the short term, but after 18 months there was no difference in comparison with those assigned to watchful waiting. Most prospective cohort studies that evaluated the association between OME and language development lacked adequate measurement of exposure or outcome, or suffered from attrition bias. Findings with regard to the association were inconsistent.

CONCLUSIONS

There is insufficient evidence to support attempts at early detection of OME in the first 4 years of life in the asymptomatic child to prevent delayed language development.
A retrospective case-control analysis of 2002 running injuries
Authors: Taunton J E , Ryan M B , Clement D B , McKenzie D C , Lloyd-Smith D R , Zumbo B D
Published: July 2002

Objective: To provide an extensive and up to date database for specific running related injuries, across the sexes, as seen at a primary care sports medicine facility, and to assess the relative risk for individual injuries based on investigation of selected risk factors.

Methods: Patient data were recorded by doctors at the Allan McGavin Sports Medicine Centre over a two year period. They included assessment of anthropometric, training, and biomechanical information. A model was constructed (with odds ratios and their 95% confidence intervals) of possible contributing factors using a dependent variable of runners with a specific injury and comparing them with a control group of runners who experienced a different injury. Variables included in the model were: height, weight, body mass index, age, activity history, weekly activity, history of injury, and calibre of runner.

Results: Most of the study group were women (54%). Some injuries occurred with a significantly higher frequency in one sex. Being less than 34 years old was reported as a risk factor across the sexes for patellofemoral pain syndrome, and in men for iliotibial band friction syndrome, patellar tendinopathy, and tibial stress syndrome. Being active for less than 8.5 years was positively associated with injury in both sexes for tibial stress syndrome; and women with a body mass index less than 21 kg/m2were at a significantly higher risk for tibial stress fractures and spinal injuries. Patellofemoral pain syndrome was the most common injury, followed by iliotibial band friction syndrome, plantar fasciitis, meniscal injuries of the knee, and tibial stress syndrome.

Conclusions: Although various risk factors were shown to be positively associated with a risk for, or protection from, specific injuries, future research should include a non-injured control group and a more precise measure of weekly running distance and running experience to validate these results.
Community based universal neonatal hearing screening by health visitors using otoacoustic emissions
Authors: Owen M , Webb M , Evans K
Published: July 2002

OBJECTIVES

To carry out a pilot study to test the feasibility of health visitors (HVs) performing neonatal otoacoustic emissions (OAE) hearing screening in the community using Echoport ILO288 and to evaluate its acceptability to parents and HVs.

DESIGN

Prospective cohort study.

SETTING

Local health centres and babies' homes in urban and rural settings in West Gloucestershire.

PARTICIPANTS

Twelve HVs, 683 babies, and their parents.

MAIN OUTCOME MEASURES

Coverage rate, age at testing, referral rate for formal audiology testing, and parental anxiety scores.

RESULTS

Of the 683 babies registered with the study HVs, 99% (675) were tested, with a median age at first test of 18 days. Parental consent for the study was refused for six of the eight not tested. Taking a unilateral pass as a screening pass (for comparison with other studies), 4% (27/675) failed the first OAE test, and 1.9% (13/675) failed a second OAE test performed by the HV within a further two weeks and were referred for formal audiology testing. One baby (0.15%) was found to have a moderate sensorineural hearing loss on brain stem auditory evoked responses, giving a false positive rate of 1.7% (12/675). Some 18% (120/675) were tested at home, of which 80% (96/120) were combined with another planned reason for HV contact. In all, 82% (555/675) of tests were carried out in health centre clinics, of which 47% (260/555) were combined purpose visits. Mean parental anxiety scores (possible range 0–5) were 0.86, 2.27, and 3.45 before the first test, first retest, and audiology test respectively. The median time taken for one HV to complete testing was 12.2 minutes (range 3–65), compared with the 15 minutes currently allocated for two HVs to perform distraction testing. Based on the results of questionnaires, the test was very well received by parents and HVs alike.

CONCLUSION

HVs are able to perform OAE testing in the neonatal period at home and in local health centre clinics. They achieve high population coverage rates and low false positive rates. Universal neonatal hearing screening by HVs using OAE testing is feasible, well received, and could be less demanding of HV time than the current distraction testing. This model of universal neonatal hearing screening should be considered by the National Screening Committee.
Decisions to treat or not to treat pneumonia in demented psychogeriatric nursing home patients: development of a guideline
Authors: van der Steen Jenny T , Muller Martien T , Ooms Marcel E , van der Wal Gerrit , Ribbe Miel W
Published: July 2002

Non-treatment decisions concerning demented patients are complex: in addition to issues concerning the health of patients, ethical and legal issues are involved. This paper describes a method for the development of a guideline that clarifies the steps to be taken in the decision making process whether to forgo curative treatment of pneumonia in psychogeriatric nursing home patients.

The method of development consisted of seven steps. Step 1 was a literature study from which ethical, juridical and medical factors concerning the patient's health and prognosis were identified. In step 2, a questionnaire was sent to 26 nursing home physicians to determine the relative importance of these factors in clinical practice. In a meeting of nine experienced physicians (step 3), the factors identified in step 2 were confirmed by most of these professionals. To prevent the final guideline being too directive, a concept guideline that included ethical and legal aspects was designed in the form of a “checklist of considerations” (step 4). Experts in the fields of nursing home medicine, ethics and law reviewed and commented on the concept guideline (step 5). The accordingly adapted “checklist of considerations” was tested in a pilot study (step 6), after which all experts endorsed the checklist (step 7).

The resulting “checklist of considerations” structures the decision making process according to three primary domains: medical aspects, patient's autonomy, and patient's best interest (see annex at end of paper).
Talking about cases in bioethics: the effect of an intensive course on health care professionals
Authors: Malek Janet I , Geller Gail , Sugarman Jeremy
Published: July 2002

Educational efforts in bioethics are prevalent, but little is known about their efficacy. Although previous work indicates that courses in bioethics have a demonstrable effect on medical students, it has not examined their effect on health care professionals. In this report, we describe a study designed to investigate the effect of bioethics education on health care professionals. At the Intensive Bioethics Course, a six-day course held annually at Georgetown University, we administered a questionnaire requiring open-ended responses to vignettes both before and after the course. Following the course, respondents defended their responses more carefully and articulated their thoughts more clearly. In addition, after the course respondents seemed to have a more subtle understanding of the relevant issues in the cases and applied theory to these cases more frequently. These findings help to formulate an understanding of the effect of bioethics education on health care professionals.
Avoiding anomalous newborns: preemptive abortion, treatment thresholds and the case of baby Messenger
Authors: Gross Michael L
Published: July 2002

In its American context the case of baby Messenger, a preterm infant disconnected from life-support by his father and allowed to die has generated debate about neonatal treatment protocols. Limited by the legal and ethical norms of the United States, this case did not consider treatment protocols that might be available in other countries such as Denmark and Israel: threshold protocols whereby certain classes of newborns are not treated, and preemptive abortion allowing one to choose late-term abortion rather than risk delivery. Each offers a viable and ethically sound avenue for dealing with the economic and social expense of anomalous newborns by aborting or not treating those most likely to burden the health care system. Objections that these protocols are antithetical to American bioethical principles are considered but rejected as each policy answers to economic justice, utility and respect for autonomy.
Commercial predictive testing: the desirability of one overseeing body
Authors: Hoedemaekers Rogeer
Published: July 2002

In Europe a process of harmonisation of standards and regulations on genetic testing has started. Public discussion and consultation are recommended, but it is not clear in every European country how the decision making process as regards the further introduction of genetic testing services should be formed. In this paper the usefulness and importance of an overseeing body for genetic screening and testing is founded on four lines of reasoning: (1) analysis of the role of value judgments in the use of the concept of (genetic) abnormality; (2) a balancing of potential benefits for all parties involved; (3) a balancing of potential disadvantages, and (4) the greater availability of commercial genetic tests in the future. It is further argued that such an overseeing body has advantages for all the interested parties.
Euthanasia—a dialogue
Authors: Berry Philip
Published: July 2002

A terminally ill man requests that his life be brought to a peaceful end by the doctor overseeing his care. The doctor, an atheist, regretfully declines. The patient, unsatisfied by the answer and increasingly desperate for relief, presses the doctor for an explanation. During the ensuing dialogue the philosophical, ethical and emotional arguments brought to bear by both the doctor and the patient are dissected.