bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Pyrene-Induced Proteins in
Mycobacterium
sp. Strain 6PY1: Evidence for Two Ring-Hydroxylating Dioxygenases
Authors: Krivobok Serge , Kuony Sylvain , Meyer Christine , Louwagie Mathilde , Willison John C. , Jouanneau Yves 
Published: June 2003

ABSTRACT

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation were investigated in the pyrene-degrading
Mycobacterium
sp. strain 6PY1. [
14
C]pyrene mineralization experiments showed that bacteria grown with either pyrene or phenanthrene produced high levels of pyrene-catabolic activity but that acetate-grown cells had no activity. As a means of identifying specific catabolic enzymes, protein extracts from bacteria grown on pyrene or on other carbon sources were analyzed by two-dimensional gel electrophoresis. Pyrene-induced proteins were tentatively identified by peptide sequence analysis. Half of them resembled enzymes known to be involved in phenanthrene degradation, with closest similarity to the corresponding enzymes from
Nocardioides
sp. strain KP7. The genes encoding the terminal components of two distinct ring-hydroxylating dioxygenases were cloned. Sequence analysis revealed that the two enzymes, designated Pdo1 and Pdo2, belong to a subfamily of dioxygenases found exclusively in gram-positive bacteria. When overproduced in
Escherichia coli
, Pdo1 and Pdo2 showed distinctive selectivities towards PAH substrates, with the former enzyme catalyzing the dihydroxylation of both pyrene and phenanthrene and the latter preferentially oxidizing phenanthrene. The catalytic activity of the Pdo2 enzyme was dramatically enhanced when electron carrier proteins of the phenanthrene dioxygenase from strain KP7 were coexpressed in recombinant cells. The Pdo2 enzyme was purified as a brown protein consisting of two types of subunits with
M
r
s of about 52,000 and 20,000. Immunoblot analysis of cell extracts from strain 6PY1 revealed that Pdo1 was present in cells grown on benzoate, phenanthrene, or pyrene and absent in acetate-grown cells. In contrast, Pdo2 could be detected only in PAH-grown cells. These results indicated that the two enzymes were differentially regulated depending on the carbon source used for growth.
NikAB- or NikB-Dependent Intracellular Recombination between Tandemly RepeatedoriTSequences of Plasmid R64 in Plasmid or Single-Stranded Phage Vectors
Authors: Furuya Nobuhisa , Komano Teruya 
Published: June 2003

ABSTRACTThe origin of transfer (oriT) of a bacterial plasmid plays a key role in both the initiation and termination of conjugative DNA transfer. We have previously shown that a conjugation-dependent recombination between the tandem R64oriTsequences cloned into pHSG398 occurred, resulting in the deletion of the intervening sequence during DNA transfer. In this study, we tandemly cloned twooriTsequences of IncI1 plasmid R64 into pUC18. Specific recombination between the twooriTsequences in pUC18 was observed withinEscherichia colicells harboring mini-R64. This recombination was found to be independent of both therecAgene and conjugative DNA transfer. The R64 genesnikAandnikB, required for conjugal DNA processing, were essential for this recombination. Although a fully active 92-bporiTsequence was required at one site for the recombination, the 44-bporiTcore sequence was sufficient at the other site. Furthermore, when twooriTsequences were tandemly cloned into the single-stranded phage vector M13 and propagated withinE. colicells, recombination between the twooriTsequences was observed, depending on thenikBgene. These results suggest that the R64 relaxase protein NikB can execute cleavage and rejoining of single-strandedoriTDNA withinE. colicells, whereas such a reaction in double-strandedoriTDNA requires collaboration of the two relaxosome proteins, NikA and NikB.
Transcriptional Regulation of the Gene Encoding an Alcohol Dehydrogenase in the Archaeon
Sulfolobus solfataricus
Involves Multiple Factors and Control Elements
Authors: Fiorentino Gabriella , Cannio Raffaele , Rossi Mosè , Bartolucci Simonetta 
Published: June 2003

ABSTRACT

A transcriptionally active region has been identified in the 5′ flanking region of the alcohol dehydrogenase gene of the crenarchaeon
Sulfolobus solfataricus
through the evaluation of the activity of putative transcriptional regulators and the role of the region upstream of the gene under specific metabolic circumstances. Electrophoretic mobility shift assays with crude extracts revealed protein complexes that most likely contain TATA box-associated factors. When the TATA element was deleted from the region, binding sites for both DNA binding proteins, such as the small chromatin structure-modeling Sso7d and Sso10b (Alba), and transcription factors, such as the repressor Lrs14, were revealed. To understand the molecular mechanisms underlying the substrate-induced expression of the
adh
gene, the promoter was analyzed for the presence of
cis
-acting elements recognized by specific transcription factors upon exposure of the cell to benzaldehyde. Progressive dissection of the identified promoter region restricted the analysis to a minimal responsive element (PAL) located immediately upstream of the transcription factor B-responsive element-TATA element, resembling typical bacterial regulatory sequences. A benzaldehyde-activated transcription factor (Bald) that specifically binds to the PAL
cis
-acting element was also identified. This protein was purified from heparin-fractionated extracts of benzaldehyde-induced cells and was shown to have a molecular mass of ∼16 kDa. The correlation between
S. solfataricus adh
gene activation and benzaldehyde-inducible occupation of a specific DNA sequence in its promoter suggests that a molecular signaling mechanism is responsible for the switch of the aromatic aldehyde metabolism as a response to environmental changes.
Bradyrhizobium japonicumNnrR, a Denitrification Regulator, Expands the FixLJ-FixK2Regulatory Cascade
Authors: Mesa Socorro , Bedmar Eulogio J. , Chanfon Astrid , Hennecke Hauke , Fischer Hans-Martin 
Published: June 2003

ABSTRACTInBradyrhizobium japonicum, a gene namednnrRwas identified which encodes a protein with high similarity to FNR/CRP-type transcriptional regulators. Mutant strains carrying annnrRnull mutation were unable to grow anaerobically in the presence of nitrate or nitrite, and they lacked both nitrate and nitrite reductase activities. Anaerobic activation of annnrR′-′lacZfusion required FixLJ and FixK2. In turn, N oxide-mediated induction ofnirandnorgenes encoding nitrite and nitric oxide reductase, respectively, depended on NnrR. Thus, NnrR expands the FixLJ-FixK2regulatory cascade by an additional control level which integrates the N oxide signal required for maximal induction of the denitrification genes.
Hepatitis B Virus Core Protein Stimulates the Proteasome-Mediated Degradation of Viral X Protein
Authors: Kim Jung-Hwan , Kang Seongman , Kim Joon , Ahn Byung-Yoon 
Published: June 2003

ABSTRACT
Hepatitis B virus (HBV) X protein (HBx) plays an essential role in viral replication and in the development of hepatocellular carcinoma. HBx has the ability to transactivate the expression of all HBV proteins, including the viral core protein HBc. Consistent with its regulatory role, HBx is relatively unstable and is present at low levels in the cell. We report here that the level of HBx was significantly reduced by the coexpression of HBc in cultured human hepatoma cells, whereas the level of HBx mRNA was unaffected. The repression of HBx by HBc was relieved by treating cells with the proteasome inhibitor MG132, indicating that HBc acts by stimulating the proteasome-mediated degradation of HBx. Moreover, the inhibitory effect of HBc was specific to HBx and did not affect other proteins, including p53, a known target of the proteasome. Although no direct physical interaction between HBc and HBx could be demonstrated, mutational analysis indicated that the C-terminal half of HBc is responsible for its inhibitory effect. These results suggest that HBc functions as a novel regulator of the HBV life cycle and of hepatocellular carcinogenesis through control of the HBx level via an inhibitory feedback type of mechanism.
Flexibility of the Adenovirus Fiber Is Required for Efficient Receptor Interaction
Authors: Wu Eugene , Pache Lars , Von Seggern Dan J. , Mullen Tina-Marie , Mikyas Yeshi , Stewart Phoebe L. , Nemerow Glen R. 
Published: June 2003

ABSTRACT
The adenovirus (Ad) fiber protein mediates Ad binding to the coxsackievirus and Ad receptor (CAR) and is thus a major determinant of viral tropism. The fiber contains three domains: an N-terminal tail that anchors the fiber to the viral capsid, a central shaft region of variable length and flexibility, and a C-terminal knob domain that binds to cell receptors. Ad type 37 (Ad37), a subgroup D virus associated with severe ocular infections, is unable to use CAR efficiently to infect host cells, despite containing a CAR binding site in its fiber knob. We hypothesized that the relatively short, inflexible Ad37 fiber protein restricts interactions with CAR at the cell surface. To test this hypothesis, we analyzed the infectivity and binding of recombinant Ad particles containing modified Ad37 or Ad5 fiber proteins. Ad5 particles equipped with a truncated Ad5 fiber or with a chimeric fiber protein comprised of the Ad5 knob fused to the short, rigid Ad37 shaft domain had significantly reduced infectivity and attachment. In contrast, placing the Ad37 knob onto the long, flexible Ad5 shaft allowed CAR-dependent virus infection and cell attachment, demonstrating the importance of the shaft domain in receptor usage. Increasing fiber rigidity by substituting the predicted flexibility modules in the Ad5 shaft with the corresponding regions of the rigid Ad37 fiber dramatically reduced both virus infection and cell attachment. Cryoelectron microscopy (cryo-EM) single-particle analysis demonstrated the increased rigidity of this chimeric fiber. These studies demonstrate that both length and flexibility of the fiber shaft regulate CAR interaction and provide a molecular explanation for the use of alternative receptors by subgroup D Ad with ocular tropism. We present a molecular model for Ad-CAR interactions at the cell surface that explains the significance of fiber flexibility in cell attachment.
Viral Escape from Dominant Simian Immunodeficiency Virus Epitope-Specific Cytotoxic T Lymphocytes in DNA-Vaccinated Rhesus Monkeys
Authors: Barouch Dan H. , Kunstman Jennifer , Glowczwskie Jennifer , Kunstman Kevin J. , Egan Michael A. , Peyerl Fred W. , Santra Sampa , Kuroda Marcelo J. , Schmitz Jörn E. , Beaudry Kristin , Krivulka Georgia R. , Lifton Michelle A. , Gorgone Darci A. , Wolinsky Steven M. , Letvin Norman L. 
Published: June 2003

ABSTRACTVirus-specific cytotoxic T lymphocytes (CTL) are critical for control of human immunodeficiency virus type 1 replication. However, viral escape from CTL recognition can undermine this immune control. Here we demonstrate the high frequency and pattern of viral escape from dominant epitope-specific CTL in SIVgagDNA-vaccinated rhesus monkeys following a heterologous simian immunodeficiency virus (SIV) challenge. DNA-vaccinated monkeys exhibited initial effective control of the SIV challenge, but this early control was lost by serial breakthroughs of viral replication over a 3-year follow-up period. Increases in plasma viral RNA correlated temporally with declines of dominant SIV epitope-specific CD8+T-lymphocyte responses and the emergence of viral mutations that escaped recognition by dominant epitope-specific CTL. Viral escape from CTL occurred in a total of seven of nine vaccinated and control monkeys, including three animals that initially controlled viral replication to undetectable levels of plasma viral RNA. These data suggest that CTL exert selective pressure on viral replication and that viral escape from CTL may be a limitation of CTL-based AIDS vaccine strategies.
The Herpes Simplex Virus Type 1 Alkaline Nuclease and Single-Stranded DNA Binding Protein Mediate Strand Exchange In Vitro
Authors: Reuven Nina Bacher , Staire Amy E. , Myers Richard S. , Weller Sandra K. 
Published: June 2003

ABSTRACT

The replication of herpes simplex virus type 1 (HSV-1) DNA is associated with a high degree of homologous recombination. While cellular enzymes may take part in mediating this recombination, we present evidence for an HSV-1-encoded recombinase activity. HSV-1 alkaline nuclease, encoded by the UL12 gene, is a 5′→3′ exonuclease that shares homology with Redα, commonly known as λ exonuclease, an exonuclease required for homologous recombination by bacteriophage lambda. The HSV-1 single-stranded DNA binding protein ICP8 is an essential protein for HSV DNA replication and possesses single-stranded DNA annealing activities like the Redβ synaptase component of the phage lambda recombinase. Here we show that UL12 and ICP8 work together to effect strand exchange much like the Red system of lambda. Purified UL12 protein and ICP8 mediated the complete exchange between a 7.25-kb M13mp18 linear double-stranded DNA molecule and circular single-stranded M13 DNA, forming a gapped circle and a displaced strand as final products. The optimal conditions for strand exchange were 1 mM MgCl
2
, 40 mM NaCl, and pH 7.5. Stoichiometric amounts of ICP8 were required, and strand exchange did not depend on the nature of the double-stranded end. Nuclease-defective UL12 could not support this reaction. These data suggest that diverse DNA viruses appear to utilize an evolutionarily conserved recombination mechanism.
Cytokine Profiles in Adult Mink Infected with Aleutian Mink Disease Parvovirus
Authors: Jensen P. V. , Castelruiz Y. , Aasted B. 
Published: June 2003

ABSTRACTThe aim of this study was to examine the levels of gamma interferon (IFN-γ)-, interleukin 4 (IL-4)-, and IL-8-producing cells in peripheral blood mononuclear cells from mink infected with the Aleutian mink disease parvovirus (ADV). As expected, ADV-infected mink developed high plasma gamma globulin values (hypergammaglobulinemia) and enhanced quantities of CD8-positive (CD8+) cells in the blood during the infection. We quantified the percentages of IFN-γ- and IL-4-positive lymphocytes and IL-8-positive monocytes up to week 38 after virus challenge. The results clearly indicated marked increases in the percentages of IFN-γ- and IL-4-producing lymphocytes during ADV infection. The total number of IL-8-producing monocytes in the blood of ADV-infected mink stayed fairly constant during the infection. In order to characterize the phenotype of the cytokine-producing cells, we performed double-labeling fluorescence-activated cell sorter (FACS) experiments with CD8 surface labeling in one channel and cytokine intracellular staining in the other. We found that most IFN-γ and IL-4 in ADV-infected mink was produced by CD8+cells, while in the uninfected mink, these cytokines were primarily produced by a cell type that was not CD8 (possibly CD4-positive cells). We also observed that IL-8 was almost exclusively produced by monocytes. All of the above findings led us to conclude that both Th1- and Th2-driven immune functions are found in mink plasmacytosis.
Functional Analysis of the Endogenous Retroviral Promoter of the Human Endothelin B Receptor Gene
Authors: Landry Josette-Renée , Mager Dixie L. 
Published: June 2003

ABSTRACT

We previously reported that the long terminal repeats (LTRs) of retroviral elements belonging to the HERV-E family contribute to the expression of the human apolipoprotein C1 (
APOC1
) and endothelin B receptor (
EDNRB
) genes by providing alternative promoters. While both LTRs were shown to promote transcription in vivo and in vitro, their respective activity and tissue specificity appeared to differ even though they shared a high degree of sequence identity. In the present study, we further characterized the promoter of the
EDNRB
LTR and delineated the regions and motifs required for strong activity. We confirmed the placenta-restricted expression of the LTR by transient transfections and quantitative real-time PCR and determined that the retroviral promoter contributes significantly to the level of
EDNRB
transcripts in placenta, where chimeric mRNAs were found to represent 15% of overall
EDNRB
mRNAs. Transient transfection of 5′ deletion constructs in cells of placental origin identified a motif, named LPE1, between positions 111 and 122 of the
EDNRB
LTR necessary for transcriptional activity. Removal of this region, which contains a putative SP1 binding site, abolished promoter activity. A second enhancing region resides between positions 175 and 215 of the LTR and was termed LPE2. Interestingly, this section contained three binding sites that were not present in the
APOC1
LTR due to minor nucleotide differences. The predicted motifs in the
EDNRB
LTR were found to likely act in symbiosis as modifications to any of the three sites reduced transcription by one-third while alterations to all three eliminated promoter activity. The results from this study illustrate how slight variations in transcriptional regulatory sequences can have a profound effect on promoter activity and demonstrate the complex regulatory effects of human endogenous retrovirus elements on human gene expression.
Poxvirus Orthologous Clusters: toward Defining the Minimum Essential Poxvirus Genome
Authors: Upton Chris , Slack Stephanie , Hunter Arwen L. , Ehlers Angelika , Roper Rachel L. 
Published: June 2003

ABSTRACT

Increasingly complex bioinformatic analysis is necessitated by the plethora of sequence information currently available. A total of 21 poxvirus genomes have now been completely sequenced and annotated, and many more genomes will be available in the next few years. First, we describe the creation of a database of continuously corrected and updated genome sequences and an easy-to-use and extremely powerful suite of software tools for the analysis of genomes, genes, and proteins. These tools are available free to all researchers and, in most cases, alleviate the need for using multiple Internet sites for analysis. Further, we describe the use of these programs to identify conserved families of genes (poxvirus orthologous clusters) and have named the software suite POCs, which is available at
www.poxvirus.org
. Using POCs, we have identified a set of 49 absolutely conserved gene families—those which are conserved between the highly diverged families of insect-infecting entomopoxviruses and vertebrate-infecting chordopoxviruses. An additional set of 41 gene families conserved in chordopoxviruses was also identified. Thus, 90 genes are completely conserved in chordopoxviruses and comprise the minimum essential genome, and these will make excellent drug, antibody, vaccine, and detection targets. Finally, we describe the use of these tools to identify necessary annotation and sequencing updates in poxvirus genomes. For example, using POCs, we identified 19 genes that were widely conserved in poxviruses but missing from the vaccinia virus strain Tian Tan 1998 GenBank file. We have reannotated and resequenced fragments of this genome and verified that these genes are conserved in Tian Tan. The results for poxvirus genes and genomes are discussed in light of evolutionary processes.
Effects of Charged Cluster Mutations on the Function of Herpes Simplex Virus Type 1 U
L
34 Protein
Authors: Bjerke Susan L. , Cowan John M. , Kerr Jelani K. , Reynolds Ashley E. , Baines Joel D. , Roller Richard J. 
Published: June 2003

ABSTRACT

Herpes simplex virus type 1 (HSV-1) is a DNA virus that acquires an envelope by budding into the inner nuclear membrane of an infected cell. Recombinant HSV-1 lacking the U
L
34 gene cannot undergo this event. U
L
34 and U
L
31, another viral protein, colocalize in an infected cell and are necessary and sufficient to target both proteins to the inner nuclear envelope. In order to define and characterize sequences of U
L
34 that are necessary for primary envelopment to occur, a library of 19 U
L
34 charged cluster mutants and a truncation mutant lacking the putative transmembrane domain (ΔTM) were generated. Mutants in this library were analyzed in a complementation assay for their ability to function in the production of infectious virus. Seven of the mutants failed to complement a U
L
34-null virus. The remainder of the mutants complemented at or near wild-type U
L
34 levels. Failure of a mutant protein to function might be the result of incorrect subcellular localization. To address this possibility, confocal microscopy was used to determine the localization of the U
L
34 protein in charged cluster mutants and ΔTM. In transfection-infection experiments, all of the functional U
L
34 mutants and four of the six noncomplementing mutants localized to the inner nuclear envelope in a manner indistinguishable from that of wild-type U
L
34. All of the noncomplementing U
L
34 mutants mediated proper localization of U
L
31. Charged clusters critical for U
L
34 function are dispersed throughout the protein sequence and do not correlate well with highly conserved regions of the protein. These data suggest that U
L
34 has at least one function in addition to mediating proper localization of U
L
31 in infected cells and provide further support for the role of U
L
34 in mediating proper localization of U
L
31 in infected cells.
High Frequency of Syncytium-Inducing and CXCR4-Tropic Viruses among Human Immunodeficiency Virus Type 1 Subtype C-Infected Patients Receiving Antiretroviral Treatment
Authors: Johnston Elizabeth R. , Zijenah Lynn S. , Mutetwa Solomon , Kantor Rami , Kittinunvorakoon Chonticha , Katzenstein David A. 
Published: June 2003

ABSTRACT
Human immunodeficiency virus type 1 (HIV-1) subtype C viruses have been found to almost exclusively use the chemokine receptor CCR5 as a coreceptor for entry, even in patients with advanced AIDS. We have characterized subtype C virus isolates from 28 patients from Harare, Zimbabwe, 20 of whom were receiving antiretroviral treatment. Virus from 10 of the treated patients induced syncytium formation (SI virus) when cultured with MT2 cells. Only non-syncytium-inducing (NSI) virus was cultured from the peripheral blood mononuclear cells of the eight patients who had not received treatment. The majority of these subtype C SI viruses were capable of using both CCR5 and CXCR4 as coreceptors for viral entry, and the consensus V3 loop sequences from the SI viruses displayed a high net charge compared to those of NSI viruses. While those on treatment had reverse transcriptase (RT) and protease mutations, there was no clear association between RT and protease drug resistance mutations and coreceptor tropism. These results suggest that CXCR4-tropic viruses are present within the quasispecies of patients infected with subtype C virus and that antiretroviral treatment may create an environment for the emergence of CXCR4 tropism.
Subchondral Bone Resorption in Temporomandibular Joint Disorders
Authors: Gruber Helen E. , Gregg John 
Published: June 2003

Several tissues are involved in temporomandibular joint (TMJ) health, including synovial fluid, the TMJ disc, articular cartilage, and subchondral bone. This article focuses upon bone resorption in temporomandibular joint disorders (TMD) and has the following objectives: (1) to provide a brief review of the current understanding of bone formation and bone resorption (bone remodeling); (2) to present selected case studies which illustrate the spectrum of bone resorption patterns in TMD patients of various ages; (3) to review previous reports in the literature describing loss of subchondral bone in TMD, and (4) to discuss the interaction between osteoporosis and TMD and the potential role for antiresorbing agents in TMD therapy.