bluuebunny/crossref_metadata_2025 huggingface dataset:
Natural Resistance to Inhibitors of the Ubiquinol Cytochrome
c
Oxidoreductase of
Rubrivivax gelatinosus
: Sequence and Functional Analysis of the Cytochrome
bc
1
Complex
Authors: Ouchane Soufian , Agalidis Ileana , Astier Chantal
Published: July 2002

Biochemical analyses of
Rubrivivax gelatinosus
membranes have revealed that the cytochrome
bc
1
complex is highly resistant to classical inhibitors including myxothiazol, stigmatellin, and antimycin. This is the first report of a strain exhibiting resistance to inhibitors of both catalytic Q
0
and Q
i
sites. Because the resistance to cytochrome
bc
1
inhibitors is primarily related to the cytochrome
b
primary structure, the
petABC
operon encoding the subunits of the cytochrome
bc
1
complex of
Rubrivivax gelatinosus
was sequenced. In addition to homologies to the corresponding proteins from other organisms, the deduced amino acid sequence of the cytochrome
b
polypeptide shows (i) an E303V substitution in the highly conserved PEWY loop involved in quinol/stigmatellin binding, (ii) other substitutions that could be involved in resistance to cytochrome
bc
1
inhibitors, and (iii) 14 residues instead of 13 between the histidines in helix IV that likely serve as the second axial ligand to the b
H
and b
L
hemes, respectively. These characteristics imply different functional properties of the cytochrome
bc
1
complex of this bacterium. The consequences of these structural features for the resistance to inhibitors and for the properties of
R. gelatinosus
cytochrome
bc
1
are discussed with reference to the structure and function of the cytochrome
bc
1
complexes from other organisms.
FtsA Mutants ofBacillus subtilisImpaired in Sporulation
Authors: Kemp Jennifer T. , Driks Adam , Losick Richard
Published: July 2002

Spore formation inBacillus subtilisinvolves a switch in the site of cell division from the midcell to a polar position. Both medial division and polar division are mediated in part by the actin-like, cytokinetic protein FtsA. We report the isolation of an FtsA mutant (FtsAD265G) that is defective in sporulation but is apparently unimpaired in vegetative growth. Sporulating cells of the mutant reach the stage of asymmetric division but are partially blocked in the subsequent morphological process of engulfment. As judged by fluorescence microscopy and electron microscopy, the FtsAD265Gmutant produces normal-looking medial septa but immature (abnormally thin) polar septa. The mutant was unimpaired in transcription under the control of Spo0A, the master regulator for entry into sporulation, but was defective in transcription under the control of σF, a regulatory protein whose activation is known to depend on polar division. An amino acid substitution at a residue (Y264) adjacent to D265 also caused a defect in sporulation. D265 and Y264 are conserved among endospore-forming bacteria, raising the possibility that these residues are involved in a sporulation-specific protein interaction that facilitates maturation of the sporulation septum and the activation of σF.
Burkholderia thailandensis
E125 Harbors a Temperate Bacteriophage Specific for
Burkholderia mallei
Authors: Woods Donald E. , Jeddeloh Jeffrey A. , Fritz David L. , DeShazer David
Published: July 2002

Burkholderia thailandensis
is a nonpathogenic gram-negative bacillus that is closely related to
Burkholderia mallei
and
Burkholderia pseudomallei
. We found that
B. thailandensis
E125 spontaneously produced a bacteriophage, termed φE125, which formed turbid plaques in top agar containing
B. mallei
ATCC 23344. We examined the host range of φE125 and found that it formed plaques on
B. mallei
but not on any other bacterial species tested, including
B. thailandensis
and
B. pseudomallei
. Examination of the bacteriophage by transmission electron microscopy revealed an isometric head and a long noncontractile tail.
B. mallei
NCTC 120 and
B. mallei
DB110795 were resistant to infection with φE125 and did not produce lipopolysaccharide (LPS) O antigen due to IS
407
A insertions in
wbiE
and
wbiG
, respectively.
wbiE
was provided in
trans
on a broad-host-range plasmid to
B. mallei
NCTC 120, and it restored LPS O-antigen production and susceptibility to φE125. The 53,373-bp φE125 genome contained 70 genes, an IS
3
family insertion sequence (IS
Bt
3), and an attachment site (
attP
) encompassing the 3′ end of a proline tRNA (UGG) gene. While the overall genetic organization of the φE125 genome was similar to λ-like bacteriophages and prophages, it also possessed a novel cluster of putative replication and lysogeny genes. The φE125 genome encoded an adenine and a cytosine methyltransferase, and purified bacteriophage DNA contained both N6-methyladenine and N4-methylcytosine. The results presented here demonstrate that φE125 is a new member of the λ supergroup of
Siphoviridae
that may be useful as a diagnostic tool for
B. mallei
.
AmtB Is Necessary for NH
4
+
-Induced Nitrogenase Switch-Off and ADP-Ribosylation in
Rhodobacter capsulatus
Authors: Yakunin Alexander F. , Hallenbeck Patrick C.
Published: July 2002

Rhodobacter capsulatus
possesses two genes potentially coding for ammonia transporters,
amtB
and
amtY
. In order to better understand their role in the physiology of this bacterium and their possible significance in nitrogen fixation, we created single-knockout mutants. Strains mutated in either
amtB
or
amtY
did not show a growth defect under any condition tested and were still capable of taking up ammonia at nearly wild-type rates, but an
amtB
mutant was no longer capable of transporting methylamine. The
amtB
strain but not the
amtY
strain was also totally defective in carrying out ADP-ribosylation of Fe-protein or the switch-off of in vivo nitrogenase activity in response to NH
4
+
addition. ADP-ribosylation in response to darkness was unaffected in
amtB
and
amtBY
strains, and glutamine synthetase activity was normally regulated in these strains in response to ammonium addition, suggesting that one role of AmtB is to function as an ammonia sensor for the processes that regulate nitrogenase activity.
The
Vibrio cholerae vieSAB
Locus Encodes a Pathway Contributing to Cholera Toxin Production
Authors: Tischler Anna D. , Lee Sang Ho , Camilli Andrew
Published: July 2002

The genes encoding cholera toxin (CT),
ctxAB
, are coregulated with those for other
Vibrio cholerae
virulence factors by a cascade of transcriptional activators, including ToxR, TcpP, and ToxT. Additional regulators that modulate expression of
ctxAB
during infection were recently identified in a genetic selection. A transposon insertion in
vieS
, the sensor kinase of the VieSAB three-component signal transduction system, resulted in failure to induce expression of a
ctxA
-recombinase fusion during murine infection. To determine which components of the VieSAB system are essential for CT regulation,
ctxAB
transcript levels were assessed by RNase protection assay in various
vieSAB
in-frame deletion mutants after growth in vitro under virulence gene inducing conditions. A threefold reduction in
ctxAB
transcript levels was observed for the Δ
vieSAB
strain; consistent with this, the Δ
vieSAB
strain produced twofold less CT protein than the wild type, and this defect was complementable in
trans
. These results suggest that the VieSAB three-component system is required for full activation of the
ctxAB
operon during in vitro growth as well as during infection. The VieSAB system may regulate
ctxAB
expression indirectly by affecting production of ToxT, because decreased
toxT
transcript levels were observed in the Δ
vieSAB
strain.
Antigen 43-Mediated Autotransporter Display, a Versatile Bacterial Cell Surface Presentation System
Authors: Kjærgaard Kristian , Hasman Henrik , Schembri Mark A. , Klemm Per
Published: July 2002

Antigen 43 (Ag43), a self-recognizing outer membrane protein ofEscherichia coli, has been converted into an efficient and versatile tool for surface display of foreign protein segments. Ag43 is an autotransporter protein characterized by the feature that all information required for transport to the outer membrane and secretion through the cell envelope is contained within the protein itself. Ag43 consists of two subunits (α and β), where the β-subunit forms an integral outer membrane translocator to which the α-subunit is noncovalently attached. The simplicity of the Ag43 system makes it ideally suited as a surface display scaffold. Here we demonstrate that the Ag43 α-module can accommodate and display correctly folded inserts and has the ability to display entire functional protein domains, exemplified by the FimH lectin domain. The presence of heterologous cysteine bridges does not interfere with surface display, and Ag43 chimeras are correctly processed into α- and β-modules, offering optional and easy release of the chimeric α-subunits. Furthermore, Ag43 can be displayed in many gram-negative bacteria. This feature is exploited for display of our chimeras in an attenuatedSalmonellastrain.
Detection of Methicillin-ResistantStaphylococcus aureusand Simultaneous Confirmation by Automated Nucleic Acid Extraction and Real-Time PCR
Authors: Grisold Andrea J. , Leitner Eva , Mühlbauer Gerhard , Marth Egon , Kessler Harald H.
Published: July 2002

A molecular assay for the simultaneous detection of aStaphylococcus aureus-specific gene and themecAgene, responsible for the resistance to methicillin in staphylococci, was evaluated. The assay included an automated DNA extraction protocol conducted with a MagNA Pure instrument and real-time PCR conducted with a LightCycler instrument. The performance and robustness of the assay were evaluated for a suspension of methicillin-resistantS. aureus(MRSA) strain with a turbidity equivalent to a McFarland standard of 0.5, which was found to be the ideal working concentration. The specificity of the new molecular assay was tested with a panel of 30 gram-negative and gram-positive bacterial strains other than MRSA. No cross-reactivity was observed. In a clinical study, 109 isolates of MRSA were investigated. All clinical MRSA isolates gave positive results for theS. aureus-specific genomic target, and all but one were positive for themecAgene. In conclusion, the new molecular assay was found to be quick, robust, and laborsaving, and it proved to be suitable for a routine molecular diagnostic laboratory.
Seroprevalence of Antibodies That React with
Anaplasma phagocytophila
, the Agent of Human Granulocytic Ehrlichiosis, in Different Populations in Westchester County, New York
Authors: Aguero-Rosenfeld Maria E. , Donnarumma Lorraine , Zentmaier Lois , Jacob Jobby , Frey Michael , Noto Richard , Carbonaro Carol A. , Wormser Gary P.
Published: July 2002

We determined the frequencies of antibodies to
Anaplasma phagocytophila
, the agent of human granulocytic ehrlichiosis (HGE), in different groups of adults and children from Westchester County, New York. The groups included 159 adult blood donors and 215 children who were seronegative for
Borrelia burgdorferi
antibodies, 118 adult patients and 57 children who were seropositive for
B. burgdorferi
antibodies, and 42 adult patients with culture-confirmed erythema migrans. Eighteen (11.3%) of the blood donors and 11 (5.1%) of the
B. burgdorferi
-seronegative children were found to have
A. phagocytophila
antibodies by indirect immunofluorescent-antibody assay (IFA). Nine of 42 (21.4%) patients with culture-confirmed erythema migrans tested at the baseline visit, 42 of 118 (35.6%) adults, and 3 of 57 (5.3%) children whose sera were reactive for
B. burgdorferi
antibodies also tested positive for
A. phagocytophila
antibodies. The geometric mean titer ranged from 219 to 315 for all groups, and the differences in titers among the groups were not statistically significant. Only one-third of the healthy blood donors reactive by IFA were confirmed to be positive by immunoblotting. We conclude that a significant proportion of adults and children without clinical evidence of HGE will test positive for
A. phagocytophila
antibodies when the conventional cutoff titer of 80 is used in the IFA. This information must be considered in interpretation of test results.
NAD-Glycohydrolase Production and
speA
and
speC
Distribution in Group A Streptococcus (GAS) Isolates Do Not Correlate with Severe GAS Diseases in the Australian Population
Authors: DelVecchio Armando , Maley Michael , Currie Bart J. , Sriprakash K. S.
Published: July 2002

Streptococcus pyogenes
isolates from a tropical region and a subtropical region of Australia with high and low incidences of severe streptococcal diseases, respectively, were analyzed for
speA
,
speB
, and
speC
gene distributions and NAD-glycohydrolase expression. No direct correlation of these characteristics with a propensity to cause invasive diseases was observed.
Impact of Cytokines on Replication in the Thymus of Primary Human Immunodeficiency Virus Type 1 Isolates from Infants
Authors: Pedroza-Martins Livia , Boscardin W. John , Anisman-Posner Deborah J. , Schols Dominique , Bryson Yvonne J. , Uittenbogaart Christel H.
Published: July 2002

Early infection of the thymus with the human immunodeficiency virus (HIV) may explain the more rapid disease progression among children infected in utero than in children infected intrapartum. Therefore, we analyzed infection of thymocytes in vitro by HIV type 1 primary isolates, obtained at or near birth, from 10 children with different disease outcomes. HIV isolates able to replicate in the thymus and impact thymopoiesis were present in all infants, regardless of the timing of viral transmission and the rate of disease progression. Isolates from newborns utilized CCR5, CXCR4, or both chemokine receptors to enter thymocytes. Viral expression was observed in discrete thymocyte subsets postinfection with HIV isolates using CXCR4 (X4) and isolates using CCR5 (R5), despite the wider distribution of CXCR4 in the thymus. In contrast to previous findings, the X4 primary isolates were not more cytopathic for thymocytes than were the R5 isolates. The cytokines interleukin-2 (IL-2), IL-4, and IL-7 increased HIV replication in the thymus by inducing differentiation and expansion of mature CD27+thymocytes expressing CXCR4 or CCR5. IL-2 and IL-4 together increased expression of CXCR4 and CCR5 in this population, whereas IL-4 and IL-7 increased CXCR4 but not CCR5 expression. IL-2 plus IL-4 increased the viral production of all pediatric isolates, but IL-4 and IL-7 had a significantly higher impact on the replication of X4 isolates compared to R5 isolates. Our studies suggest that coreceptor use by HIV primary isolates is important but is not the sole determinant of HIV pathogenesis in the thymus.
The Lipopolysaccharide and β-1,3-Glucan Binding Protein Gene Is Upregulated in White Spot Virus-Infected Shrimp (Penaeus stylirostris)
Authors: Roux Michelle M. , Pain Arnab , Klimpel Kurt R. , Dhar Arun K.
Published: July 2002

Pattern recognition proteins such as lipopolysaccharide and β-1,3-glucan binding protein (LGBP) play an important role in the innate immune response of crustaceans and insects. Random sequencing of cDNA clones from a hepatopancreas cDNA library of white spot virus (WSV)-infected shrimp provided a partial cDNA (PsEST-289) that showed similarity to the LGBP gene of crayfish and insects. Subsequently full-length cDNA was cloned by the 5′-RACE (rapid amplification of cDNA ends) technique and sequenced. The shrimp LGBP gene is 1,352 bases in length and is capable of encoding a polypeptide of 376 amino acids that showed significant similarity to homologous genes from crayfish, insects, earthworms, and sea urchins. Analysis of the shrimp LGBP deduced amino acid sequence identified conserved features of this gene family including a potential recognition motif for β-(1→3) linkage of polysaccharides and putative RGD cell adhesion sites. It is known that LGBP gene expression is upregulated in bacterial and fungal infection and that the binding of lipopolysaccharide and β-1,3-glucan to LGBP activates the prophenoloxidase (proPO) cascade. The temporal expression of LGBP and proPO genes in healthy and WSV-challengedPenaeus stylirostrisshrimp was measured by real-time quantitative reverse transcription-PCR, and we showed that LGBP gene expression in shrimp was upregulated as the WSV infection progressed. Interestingly, the proPO expression was upregulated initially after infection followed by a downregulation as the viral infection progressed. The downward trend in the expression of proPO coincided with the detection of WSV in the infected shrimp. Our data suggest that shrimp LGBP is an inducible acute-phase protein that may play a critical role in shrimp-WSV interaction and that the WSV infection regulates the activation and/or activity of the proPO cascade in a novel way.
Amino Acid Substitutions in the V Domain of Nectin-1 (HveC) That Impair Entry Activity for Herpes Simplex Virus Types 1 and 2 but Not for Pseudorabies Virus or Bovine Herpesvirus 1
Authors: Martinez Wanda M. , Spear Patricia G.
Published: July 2002

The entry of herpes simplex virus (HSV) into cells requires the interaction of viral glycoprotein D (gD) with a cellular gD receptor to trigger the fusion of viral and cellular membranes. Nectin-1, a member of the immunoglobulin superfamily, can serve as a gD receptor for HSV types 1 and 2 (HSV-1 and HSV-2, respectively) as well as for the animal herpesviruses porcine pseudorabies virus (PRV) and bovine herpesvirus 1 (BHV-1). The HSV-1 gD binding domain of nectin-1 is hypothesized to overlap amino acids 64 to 104 of the N-terminal variable domain-like immunoglobulin domain. Moreover, the HSV-1 and PRV gDs compete for binding to nectin-1. Here we report that two amino acids within this region, at positions 77 and 85, are critical for HSV-1 and HSV-2 entry but not for the entry of PRV or BHV-1. Replacement of either amino acid 77 or amino acid 85 reduced HSV-1 and HSV-2 gD binding but had a lesser effect on HSV entry activity, suggesting that weak interactions between gD and nectin-1 are sufficient to trigger the mechanism of HSV entry. Substitution of both amino acid 77 and amino acid 85 in nectin-1 significantly impaired entry activity for HSV-1 and HSV-2 and eliminated binding to soluble forms of HSV-1 and HSV-2 gDs but did not impair the entry of PRV and BHV-1. Thus, amino acids 77 and 85 of nectin-1 form part of the interface with HSV gD or influence the conformation of that interface. Moreover, the binding sites for HSV and PRV or BHV-1 gDs on nectin-1 may overlap but are not identical.
pH Reduction as a Trigger for Dissociation of Herpes Simplex Virus Type 1 Scaffolds
Authors: McClelland David A. , Aitken James D. , Bhella David , McNab David , Mitchell Joyce , Kelly Sharon M. , Price Nicholas C. , Rixon Frazer J.
Published: July 2002

Assembly of the infectious herpes simplex virus type 1 virion is a complex, multistage process that begins with the production of a procapsid, which is formed by the condensation of capsid shell proteins around an internal scaffold fashioned from multiple copies of the scaffolding protein, pre-VP22a. The ability of pre-VP22a to interact with itself is an essential feature of this process. However, this self-interaction must subsequently be reversed to allow the scaffolding proteins to exit from the capsid to make room for the viral genome to be packaged. The nature of the process by which dissociation of the scaffold is accomplished is unknown. Therefore, to investigate this process, the properties of isolated scaffold particles were investigated. Electron microscopy and gradient sedimentation studies showed that the particles could be dissociated by low concentrations of chaotropic agents and by moderate reductions in pH (from 7.2 to 5.5). Fluorescence spectroscopy and circular dichroism analyses revealed that there was relatively little change in tertiary and secondary structures under these conditions, indicating that major structural transformations are not required for the dissociation process. We suggest the possibility that dissociation of the scaffold may be triggered by a reduction in pH brought about by the entry of the viral DNA into the capsid.
Hepatitis C Virus Subgenomic Replicons Induce Endoplasmic Reticulum Stress Activating an Intracellular Signaling Pathway
Authors: Tardif Keith D. , Mori Kazutoshi , Siddiqui Aleem
Published: July 2002

Hepatitis C virus (HCV) replicates from a ribonucleoprotein (RNP) complex that is associated with the endoplasmic reticulum (ER) membrane. The replication activities of the HCV subgenomic replicon are shown here to induce ER stress. In response to this stress, cells expressing HCV replicons induce the unfolded protein response (UPR), an ER-to-nucleus intracellular signaling pathway. The UPR is initiated by the proteolytic cleavage of a transmembrane protein, ATF6. The resulting cytoplasmic protein fragment of ATF6 functions as a transcription factor in the nucleus and activates selective genes required for an ER stress response. ATF6 activation leads to increased transcriptional levels of GRP78, an ER luminal chaperone protein. However, the overall level of GRP78 protein is decreased. While ER stress is also known to affect translational attenuation, cells expressing HCV replicons have lower levels of phosphorylation of the α subunit of eukaryotic initiation factor 2. Interestingly, cap-independent internal ribosome entry site-mediated translation directed by the 5′ noncoding region of HCV and GRP78 is activated in cells expressing HCV replicons. These studies provide insight into the effects of HCV replication on intracellular events and the mechanisms underlying liver pathogenesis.
Mutation of the Catalytic Domain of the Foamy Virus Reverse Transcriptase Leads to Loss of Processivity and Infectivity
Authors: Rinke Carolyn S. , Boyer Paul L. , Sullivan Mark D. , Hughes Stephen H. , Linial Maxine L.
Published: July 2002

Foamy virus (FV) replication is resistant to most nucleoside analog reverse transcriptase (RT) inhibitors. In an attempt to create a 2′,3′-dideoxy-3′-thiacytidine (3TC)-sensitive virus, the second residue in the highly conserved YXDD motif of simian foamy virus-chimpanzee (human isolate) [SFVcpz(hu)] RT was changed from Val (V) to Met (M). Unexpectedly, the resultant virus, SFVcpz(hu) RT-V313M, replicated poorly, and Met rapidly reverted to Val. Despite the presence of approximately 50% of wild-type RT activity in RT-V313M virions, full-length DNA products were not detected in transfected cells. Using purified recombinant enzymes, we found that the wild-type FV RT is significantly more processive than human immunodeficiency virus type 1 RT. However, the V313M mutant has about 40% of the wild-type level of FV RT activity and has a lower processivity than the wild-type FV enzyme. The V313M mutant RT is also relatively resistant to 3TC. These results suggest that the decrease in RT activity and processivity of FV RT-V313M prevents completion of reverse transcription and greatly diminishes viral replication.
Analysis of Antigenicity and Topology of E2 Glycoprotein Present on Recombinant Hepatitis C Virus-Like Particles
Authors: Clayton Reginald F. , Owsianka Ania , Aitken Jim , Graham Susan , Bhella David , Patel Arvind H.
Published: July 2002

Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2
660
) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2
660
with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2
660
but not VLPs or FL E1E2 complex. The antigenic differences between E2
660,
FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.
Differential Transmission of Human Immunodeficiency Virus Type 1 by Distinct Subsets of Effector Dendritic Cells
Authors: Sanders Rogier W. , de Jong Esther C. , Baldwin Christopher E. , Schuitemaker Joost H. N. , Kapsenberg Martien L. , Berkhout Ben
Published: July 2002

Dendritic cells (DC) support human immunodeficiency virus type 1 (HIV-1) transmission by capture of the virus particle in the mucosa and subsequent transport to the draining lymph node, where HIV-1 is presented to CD4+Th cells. Virus transmission involves a high-affinity interaction between the DC-specific surface molecule DC-SIGN and the viral envelope glycoprotein gp120 and subsequent internalization of the virus, which remains infectious. The mechanism of viral transmission from DC to T cells is currently unknown. Sentinel immature DC (iDC) develop into Th1-promoting effector DC1 or Th2-promoting DC2, depending on the activation signals. We studied the ability of these effector DC subsets to support HIV-1 transmission in vitro. Compared with iDC, virus transmission is greatly upregulated for the DC1 subset, whereas DC2 cells are inactive. Increased transmission by DC1 correlates with increased expression of ICAM-1, and blocking studies confirm that ICAM-1 expression on DC is important for HIV transmission. The ICAM-1-LFA-1 interaction is known to be important for immunological cross talk between DC and T cells, and our results indicate that this cell-cell contact is exploited by HIV-1 for efficient transmission.
Pharmacological Cyclin-Dependent Kinase Inhibitors Inhibit Replication of Wild-Type and Drug-Resistant Strains of Herpes Simplex Virus and Human Immunodeficiency Virus Type 1 by Targeting Cellular, Not Viral, Proteins
Authors: Schang Luis M. , Bantly Andrew , Knockaert Marie , Shaheen Farida , Meijer Laurent , Malim Michael H. , Gray Nathanael S. , Schaffer Priscilla A.
Published: July 2002

Pharmacological cyclin-dependent kinase (cdk) inhibitors (PCIs) block replication of several viruses, including herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus type 1 (HIV-1). Yet, these antiviral effects could result from inhibition of either cellular cdks or viral enzymes. For example, in addition to cellular cdks, PCIs could inhibit any of the herpesvirus-encoded kinases, DNA replication proteins, or proteins involved in nucleotide metabolism. To address this issue, we asked whether purine-derived PCIs (P-PCIs) inhibit HSV and HIV-1 replication by targeting cellular or viral proteins. P-PCIs inhibited replication of HSV-1 and -2 and HIV-1, which require cellular cdks to replicate, but not vaccinia virus or lymphocytic choriomeningitis virus, which are not known to require cdks to replicate. P-PCIs also inhibited strains of HSV-1 and HIV-1 that are resistant to conventional antiviral drugs, which target viral proteins. In addition, the anti-HSV effects of P-PCIs and a conventional antiherpesvirus drug, acyclovir, were additive, demonstrating that the two drugs act by distinct mechanisms. Lastly, the spectrum of proteins that bound to P-PCIs in extracts of mock- and HSV-infected cells was the same. Based on these observations, we conclude that P-PCIs inhibit virus replication by targeting cellular, not viral, proteins.
Bunyamwera Bunyavirus Nonstructural Protein NSs Counteracts the Induction of Alpha/Beta Interferon
Authors: Weber Friedemann , Bridgen Anne , Fazakerley John K. , Streitenfeld Hein , Kessler Nina , Randall Richard E. , Elliott Richard M.
Published: July 2002

Production of alpha/beta interferons (IFN-α/β) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many viruses therefore encode factors that subvert the IFN system to enhance their virulence.
Bunyamwera virus
(BUN) is the prototype of the
Bunyaviridae
family. By using reverse genetics, we previously produced a recombinant virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-α/β, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-κB and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-3. Furthermore, both in cultured cells and in mice lacking a functional IFN-α/β system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-α/β in order to increase the virulence of Bunyamwera virus.
Role for Macrophage Inflammatory Protein 2 (MIP-2), MIP-1α, and Interleukin-1α in the Delayed-Type Hypersensitivity Response to Viral Antigen
Authors: Tumpey Terrence M. , Fenton Robin , Molesworth-Kenyon Sara , Oakes John E. , Lausch Robert N.
Published: July 2002

BALB/c mice sensitized to herpes simplex virus type 1 (HSV-1) develop a vigorous delayed-type hypersensitivity (DTH) response upon intradermal virus antigen challenge. Although CD4+T cells are a key mediator of this response, neutrophils are the most abundant cells at the antigen challenge site both initially and at the peak of the reaction. We investigated what role, if any, neutrophils play in the DTH to a viral antigen. We show here that antibody-mediated depletion of neutrophils 1 day before antigen challenge significantly suppressed ear swelling and markedly reduced cellular influx. Additionally, neutrophil depletion was associated with decreased expression of macrophage inflammatory protein 2 (MIP-2) and MIP-1α, as well as with a >60-fold increase in HSV-1 replication. Neutralizing antibodies to neutrophil chemoattractants MIP-2 or MIP-1α but not KC significantly suppressed DTH and sharply reduced neutrophil accumulation in the ear pinna. Purified bone marrow-derived neutrophils exposed to interleukin-1α (IL-1α) produced chemokines in an 8-h assay. Administration of neutralizing antibody to IL-1α significantly reduced ear swelling and suppressed the levels of MIP-2, MIP-1α, MIP-1β, and RANTES. We conclude that neutrophils are a critical component of the DTH response to viral antigen. They are recruited to the DTH test site by MIP-2 and MIP-1α, where they can be activated by IL-1α. The infiltrating cells also help suppress virus replication in immunized mice.
Inhibition of Chemokine Expression by Adenovirus Early Region Three (E3) Genes
Authors: Lesokhin Alexander M. , Delgado-Lopez Fernando , Horwitz Marshall S.
Published: July 2002

Adenoviruses (Ad) have a variety of immunoregulatory genes, many of which are clustered in a 3.5-kb segment of DNA known as early region 3 (E3). Ad E3 codes for proteins that downregulate surface expression of class I major histocompatibility antigens and also inhibit tumor necrosis factor alpha (TNF-α)- and Fas-induced cytolysis. We were interested in determining whether chemokine production or activity might also be inhibited by Ad E3 and we have studied this function in a human astrocytoma cell line, U373. Astrocytes constitute a part of the blood-brain barrier, and chemokines (IP-10, IL-8, MCP-1-4, and MIPs) expressed by them may contribute to leukocyte infiltration within the brain during inflammation. When U373 cells are activated by the proinflammatory molecule TNF-α, the increase in chemokine MCP-1, IL-8, and IP-10 transcripts is blocked by a recombinant Ad expressing the E3 genes under cytomegalovirus promoter control. Comparable Ads expressing green fluorescent protein in place of E3 have no effect on these chemokines. Ads also have been extensively studied as gene therapy vectors and most have a deletion of the E3 region to permit the insertion of larger fragments of foreign DNA. Our results suggest that construction of Ad vectors to include E3 expression cassettes will improve the efficacy and safety of such viral-based gene therapy protocols.
Heparan Sulfate Mediates Infection of High-Neurovirulence Theiler's Viruses
Authors: Reddi Honey V. , Lipton Howard L.
Published: July 2002

The mechanisms by which Theiler's murine encephalomyelitis virus (TMEV) binds and enters host cells and the molecules involved are not completely understood. In this study, we demonstrate that the high-neurovirulence TMEV GDVII virus uses the glycosaminoglycan heparan sulfate (HS) as an attachment factor that is required for efficient infection. Studies based on soluble HS-mediated inhibition of attachment and infection, removal of HS with specific enzymes, and blocking with anti-HS antibodies establish that HS mediates GDVII virus entry into mammalian cells. Data from defined proteoglycan-deficient Chinese hamster ovary mutant cells further support the role of HS in GDVII infection and indicate that the extent of sulfation is critical for infection. Neuraminidase treatment of proteoglycan-deficient cells restores permissiveness to GDVII virus, indicating that sialic acid hinders direct access of virus to the protein entry receptor. A model of the potential steps in GDVII virus entry into mammalian cells involving HS is proposed.
The Relationship between Simian Immunodeficiency Virus RNA Levels and the mRNA Levels of Alpha/Beta Interferons (IFN-α/β) and IFN-α/β-Inducible Mx in Lymphoid Tissues of Rhesus Macaques during Acute and Chronic Infection
Authors: Abel Kristina , Alegria-Hartman Michelle J. , Rothaeusler Kristina , Marthas Marta , Miller Christopher J.
Published: July 2002

To define the role of alpha/beta interferons (IFN-α/β) in simian immunodeficiency virus (SIV) infection, IFN-α and IFN-β mRNA levels and mRNA levels of Mx, an antiviral effector molecule, were determined in lymphoid tissues of rhesus macaques infected with pathogenic SIV. IFN-α/β responses were induced during the acute phase and persisted in various lymphoid tissues throughout the chronic phase of infection. IFN-α/β responses were most consistent in tissues with high viral RNA levels; thus, IFN-α/β responses were not generally associated with effective control of SIV replication. IFN-α/β responses were differentially regulated in different lymphoid tissues and at different stages of infection. The most consistent IFN-α/β responses in acute and chronic SIV infection were observed in peripheral lymph nodes. In the spleen, only a transient increase in IFN-α/β mRNA levels during acute SIV infection was observed. Further, IFN-α and IFN-β mRNA levels showed a tissue-specific expression pattern during the chronic, but not the acute, phase of infection. In the acute phase of infection, SIV RNA levels in lymphoid tissues of rhesus macaques correlated with mRNA levels of both IFN-α and IFN-β, whereas during chronic SIV infection only increased IFN-α mRNA levels correlated with the level of virus replication in the same tissues. In lymphoid tissues of all SIV-infected monkeys, higher viral RNA levels were associated with increased Mx mRNA levels. We found no evidence that monkeys with increased Mx mRNA levels in lymphoid tissues had enhanced control of virus replication. In fact, Mx mRNA levels were associated with high viral RNA levels in lymphoid tissues of chronically infected animals.
Recovery of Virulent and RNase-Negative Attenuated Type 2 Bovine Viral Diarrhea Viruses from Infectious cDNA Clones
Authors: Meyer Christiane , von Freyburg Martina , Elbers Knut , Meyers Gregor
Published: July 2002

Cloned cDNA derived from the genome of the virulent type 2 bovine viral diarrhea virus (BVDV) strain NY'93/C was sequenced and served for establishment of the infectious cDNA clone pKANE40A. Virus recovered from pKANE40A exhibited growth characteristics similar to those of wild-type BVDV NY'93/C and proved to be clinically indistinguishable from the wild-type virus in animal experiments. A virus mutant in which the RNase residing in the viral glycoprotein Ernswas inactivated, revealed an attenuated phenotype. The plasmid pKANE40A represents the first infectious cDNA clone established for a type 2 BVDV and offers a variety of new approaches to analyze the mechanisms of BVDV-induced disease in cattle.
9-Isothiocyanatoanthracene as a Versatile Starting Compound in the Chemistry of Anthracen-9-yl Derivatives
Authors: Janovec Ladislav , Suchár Gejza , Imrich Ján , Kristian Pavol , Sasinková Vlasta , Alföldi Juraj , Sedlák Erik
Published: July 2002

The synthesis, structure and reactivity of 9-isothiocyanatoanthracene (5) likewise the conformation of the corresponding thioureas (6a-6i) have been studied. The 13C NMR substituent chemical shift values of the NCS group and correlations between spectral parameters (IR, NMR) and rate constans of reactions of 5, acridin-9-yl (14), phenyl (15) and benzoyl isothiocyanate (16) with butylamine were found. Significant increase of the fluorescence of the synthesized anthracenylthioureas with amino acid rests refers to the possibility to use 5 as potential biomarker.
Halogenation of Tris(amido)tantalacarboranes with Dihalomethanes CH2X2 (X = Cl, Br)
Authors: Fox Mark A. , Goeta Andrés E. , Hughes Andrew K. , Malget John M. , Wade Ken
Published: July 2002

Slow reactions of isomeric metallacarboranes of general formulae [(NMe2)3TaC2B9H11] (3 isomers) and [(NMe2)3TaC2B9H10Me] (3 isomers) with CD2Cl2 afford quantitative yields of monochloro complexes [Cl(NMe2)2TaC2B9H11] and [Cl(NMe2)2TaC2B9H10Me]. Exposure to CD2Cl2 for months leads to solutions containing about 70% of the dichlorides in three cases. More prolonged exposure of these and the other monochlorides leads to a mixture of boron-substituted complexes. Hydrolysis of [3,3,3-(NMe2)3-3,1,2-TaC2B9H11] by moist toluene results in the formation of the oxo-bridged complex 3,3'-[3,3-(NMe2)2-3,1,2-TaC2B9H11]2(μ-O), characterised by single-crystal X-ray crystallography. The limited solubility of the latter complex in CD2Cl2 eliminates the presence of this compound in the reaction of [3,3,3-(NMe2)3-3,1,2-TaC2B9H11] with CD2Cl2. The reaction of [2,2,2-(NMe2)3-2,1,12-TaC2B9H11] with CH2Br2 in C6D6 quantitatively yields the monobromide [2-Br-2,2-(NMe2)2-2,1,12-TaC2B9H11]. Prolonged reaction with CH2Br2 leads directly to isomeric boron-substituted complexes with no evidence for dibromides. The influence on 11B, 13C and 1H NMR chemical shifts of replacing an amide group in [(NMe2)3TaC2B9H11] with chloride to give [Cl(NMe2)2TaC2B9H11] is also discussed.
Cancer incidence and magnetic field exposure in industries using resistance welding in Sweden
Authors: Håkansson N , Floderus B , Gustavsson P , Johansen C , Olsen J H
Published: July 2002

Aims:To investigate cancer incidence in workers exposed to high levels of extremely low frequency magnetic fields (ELF-MF).

Methods:A cohort based on the engineering industry was established. Industries assumed to use resistance welding in production were chosen in order to increase the prevalence of high exposed subjects and to reduce the influence of confounding factors. All men and women employed in these branches during 1985–94 were selected, 537 692 men and 180 529 women. Occupation, based on census information from 1980, 1985, and 1990, was linked to a job exposure matrix on ELF-MF. Four exposure groups were used by stratifying on mean workday ELF-MF exposure, using the lowest exposure group as reference. Cancer incidence was obtained by linkage to the Swedish Cancer Registry.

Results:Men in the very high exposure group showed an increased incidence of tumours of the kidney, pituitary gland, and biliary passages and liver; for these cancer sites an exposure–response relation was indicated. Women in the very high exposure group showed an increased incidence of astrocytoma I–IV, with a clear exposure–response pattern. An association was suggested in the high exposure group only, for cancer of the corpus uteri and multiple myeloma. Decreased risks in the very high exposure group among men were found for cancer of the colon and connective tissue/muscle.

Conclusions:The results on cancer of the liver, kidney, and pituitary gland among men are in accordance with previous observations. Regarding brain tumours and leukaemia, the outcome for women provided further support of an association. The hypothesis of a biological mechanism involving the endocrine system was partly supported.
Influence of thrombolytic therapy on the patterns of ventricular septal rupture after acute myocardial infarction
Authors: Rhydwen G R , Charman S , Schofield P M
Published: July 2002

Background

Post-myocardial infarction ventricular septal defect (VSD) complicates ∼2% of myocardial infarctions. Thrombolytic therapy may accelerate the time from myocardial infarction to VSD formation. The effects of thrombolytic therapy in patients with a post-myocardial infarction VSD were investigated.

Method

Demographic, procedural, and event data were retrospectively analysed in patients transferred to a regional cardiothoracic centre with the diagnosis of post-myocardial infarction VSD over five years.

Results

Twenty nine patients were analysed; 15 received thrombolytic therapy: 10 (<12 hours) early and five (≥12 hours) late. The median time to post-myocardial infarction VSD was shorter with thrombolytic therapy at 1 v 5.5 days (p=0.01). The median time to post-myocardial infarction VSD was shorter with early compared with late thrombolytic therapy at 1 v 6 days (p<0.01). There was no difference between late and no thrombolytic therapy, 5.5 v 6 days. Patients treated with thrombolytic therapy had a trend towards higher mortality at 11/15 (73%) compared with 5/14 (36%) (p=0.066). Twenty five (86%) patients had surgery. All four not having surgery died. Surgical survival was 13/25 (52%) at discharge and six months of follow up. Within the surgical group survival with prior thrombolytic therapy was 4/25 (25%) and 9/13 (69%) without (p=0.07).

Conclusion

There appears to be an earlier presentation of post-myocardial infarction VSD when thrombolytic therapy has been used. An early presentation can carry a worse prognosis and may have implications for the identification and treatment of this life threatening complication.
Population structure of the endangered wood decay fungus Phellinus nigrolimitatus (Basidiomycota)
Authors: Kauserud Håvard , Schumacher Trond
Published: July 2002

The population structure of five Fennoscandian geographic populations of the endangered wood-decay fungus Phellinus nigrolimitatus (Romell) Bourdot et Galzin was examined by analyses of nuclear ribosomal DNA (nrDNA) spacer sequences (ITS and IGS1) and a partial sequence of the elongation factor 1α gene (efa). A high level of sequence variation was observed in ITS and IGS1, suggesting restrictions in nrDNA homogenization in this taxon. Six polymerase chain reaction  restriction fragment length polymorphism (PCR-RFLP) markers, five located in nrDNA and one in efa, suggest that the geographic populations are genetically very similar, presumably owing to recent gene flow. However, linkage disequilibria were obtained in 50% of the cases in tests between the five nrDNA PCR-RFLP markers. The calculated FST values from the linked nrDNA markers and the unlinked efa marker were congruent, ranging from 0.006 to 0.042. In one geographic population, the efa locus showed significant deviation from Hardy Weinberg expectations. Somatic incompatibility tests demonstrated that isolates derived from different basidiocarps and different logs belonged to different genets. In a microscale study including three logs, the independent assays of PCR-RFLP analysis and somatic incompatibility tests distinguished 10 genets. Life history traits and conservation status of P. nigrolimitatus are discussed in light of the results.Key words: Phellinus nigrolimitatus, population structure, somatic incompatibility, PCR-RFLP, nrDNA.
Dormancy in peach (Prunus persica) flower buds. V. Anatomy of bud development in relation to phenological stage
Authors: Reinoso Herminda , Luna Virginia , Pharis Richard P , Bottini Rubén
Published: July 2002

Anatomical changes in the peach (Prunus persica (L.) Batsch.) flower buds were defined and then assessed and correlated with the phenological stage from early dormancy through to flower opening. The peach flower bud, unlike the vegetative bud, shows a continuous anatomical development during the late autumn and winter dormancy period, even though there are no obvious macroscopic changes. Sterile whorls differentiate rapidly in late summer through early autumn. In contrast, fertile whorls develop very slowly during winter; their rapid development begins in late winter and continues through early spring. The androecium develops throughout the winter, while the gynoecium develops in late winter. By late winter, the anthers begin to undergo microsporogenesis and microgametogenesis and the ovaries have formed ovules. Vascular connections between flower primordia and branch wood are complete by late winter, when rapid phenological changes begin. At this point in time, the peach floral bud enters a "rapid maturation phase" that ends in flower opening. Thus, for the peach flower bud at least, the concept of dormancy as "a temporary suspension of visible growth of any plant structure containing a meristem" that was proposed by earlier researchers appears inappropriate. Rather, cell division, enlargement, and differentiation, which lead to organogenesis, take place throughout the entire "dormancy" period.Key words: dormancy, floral bud anatomy, floral bud phenology, peach, Prunus persica.
Magnetostratigraphic and biostratigraphic correlation of late Campanian and Maastrichtian marine and continental strata from the Red Deer Valley to the Cypress Hills, Alberta, Canada
Authors: Lerbekmo J F , Braman D R
Published: July 2002

Two stratigraphic sequences, Red Deer Valley and Cypress Hills sections in Alberta, spanning the late Campanian to Paleocene are magnetostratigraphically and biostratigraphically characterized. The Red Deer Valley section had previously been studied, but additional material has allowed the extension of the magnetozonation downwards into the Bearpaw Formation. Higher sample resolution has allowed the identification of a number of magnetosubzones in 32n, 32r, and 33n. Four reversed magnetosubzones have been identified in magnetozone 32n in addition to the one presently recognized in geomagnetic polarity time scales. One normal subzone in 32r and one reversed subzone in 33n have also been recorded. The 32r33n boundary is shown to occur in the Baculites cuneatus Zone. The Campanian-Maastrichtian boundary is indicated to be at or near the Baculites eliasi  Baculites baculus boundary at the top of 32n. Comparisons of the biostratigraphy between the two areas indicate that palynomorph ranges differ from data published to date, suggesting that readjustments to the previously published zonation are needed. Correlations between the two areas are possible using the magnetostratigraphic and palynological zones and allow comparisons of depositional rates.