bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vitro Evaluation of the Effectiveness of the Macrolide Rokitamycin and Chlorpromazine against
Acanthamoeba castellanii
Authors: Mattana A. , Biancu G. , Alberti L. , Accardo A. , Delogu G. , Fiori P. L. , Cappuccinelli P. 
Published: November 2004

ABSTRACT

The present study demonstrates the in vitro effectiveness of the macrolide rokitamycin and the phenothiazine compound chlorpromazine against
Acanthamoeba castellanii
. Growth curve evaluations revealed that both drugs inhibit trophozoite growth in dose- and time-dependent ways. The effects of both drugs when they were used at the MICs at which 100% of isolates are inhibited were amoebistatic, but at higher doses they were amoebicidal as well as cysticidal. Experiments showed that when rokitamycin was associated with chlorpromazine or amphotericin B, rokitamycin enhanced their activities. Furthermore, low doses of rokitamycin and chlorpromazine, alone or in combination, blocked the cytopathic effect of
A. castellanii
against WKD cells derived from the human cornea. These results may have important significance in the development of new anti-
Acanthamoeba
compounds.
Gln145Met/Leu Changes in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confer Resistance to Nucleoside and Nonnucleoside Analogs and Impair Virus Replication
Authors: Paolucci Stefania , Baldanti Fausto , Maga Giovanni , Cancio Reynel , Zazzi Maurizio , Zavattoni Maurizio , Chiesa Antonella , Spadari Silvio , Gerna Giuseppe 
Published: November 2004

ABSTRACTThe frequencies of multidrug resistance-associated mutations at codons 145, 151, and 69 of the human immunodeficiency virus (HIV) reverse transcriptase (RT) gene in strains from a group of 3,595 highly active antiretroviral therapy (HAART)-experienced patients were 0.22, 2.36, and 0.86%, respectively. Several amino acid substitutions different from the recently reported Gln145Met change (S. Paolucci, F. Baldanti, M. Tinelli, G. Maga, and G. Gerna, AIDS 17:924-927, 2003) were detected at position 145. Thus, amino acid substitutions selected at position 145 were introduced into the wild-type HIV type 1 (HIV-1) RT gene by site-directed mutagenesis, and recombinant HIV strains were assayed for their drug susceptibilities. Only Met and Leu substitutions at position 145 of the HIV-1 RT conferred multidrug resistance, while other amino acid changes did not. Lower levels of replication of the Gln145Met recombinant strain compared with those of both Gln151Met and wild-type recombinant strains were observed. In in vitro inhibition assays, expression and purification of the recombinant Gln145Met HIV-1 RT revealed a strong loss of catalytic efficiency of the mutated enzyme, as well as significant resistance to both zidovudine and efavirenz. Specific amino acid substitutions in the HIV RT nucleotide-binding pocket might affect both antiretroviral drug recognition and binding and decrease the level of virus replication, possibly by interfering with the enzyme activity. This finding may explain the lower frequency of Gln145Met/Leu mutations observed compared with the frequencies of Gln151Met/Leu mutations and the insertion at position 69 in HAART-experienced patients.
Functional Cloning of
Bacillus anthracis
Dihydrofolate Reductase and Confirmation of Natural Resistance to Trimethoprim
Authors: Barrow Esther W. , Bourne Philip C. , Barrow William W. 
Published: November 2004

ABSTRACT

Bacillus anthracis
is reported to be naturally resistant to trimethoprim (TMP), a drug that inhibits dihydrofolate reductase (DHFR), a key enzyme in the folate pathway. A microdilution broth assay established that the MIC of TMP for
B. anthracis
Sterne is >2,048 but ≤4,096 μg/ml. A putative DHFR sequence was amplified from
B. anthracis
Sterne genomic DNA. The PCR product was cloned into the Invitrogen pCRT7/CT-TOPO vector, followed by transformation into
Escherichia coli
TOP10F′ chemically competent cells. Plasmid DNA from a clone showing the correct construct with a thrombin cleavage site attached downstream from the terminus of the cloned PCR product was transformed into
E. coli
BL21 Star (DE3)pLysS competent cells for expression of the six-histidine-tagged fusion protein and purification on a His-Bind resin column. Functionality of the purified Sterne recombinant DHFR (Sterne rDHFR) was confirmed in an established enzyme assay. The 50% inhibitory concentrations of TMP and methotrexate for the Sterne rDHFR were found to be 77,233 and 12.2 nM, respectively. TMP resistance was observed with
E. coli
BL21 Star (DE3)pLysS competent cells transformed with the Sterne DHFR gene. Alignment of the amino acid sequence of the Sterne DHFR gene revealed 100% homology with various virulent strains of
B. anthracis
. These results confirm the natural resistance of
B. anthracis
to TMP and clarify that the resistance is correlated to a lack of selectivity for the chromosomally encoded gene product. These findings will assist in the development of narrow-spectrum antimicrobial agents for treatment of anthrax.
Molecular Characterization of a β-Lactamase Gene,
bla
GIM-1
, Encoding a New Subclass of Metallo-β-Lactamase
Authors: Castanheira Mariana , Toleman Mark A. , Jones Ronald N. , Schmidt Franz J. , Walsh Timothy R. 
Published: November 2004

ABSTRACT

As part of the SENTRY Antimicrobial Surveillance Program in 2002, five multidrug-resistant
Pseudomonas aeruginosa
clinical isolates were detected with metallo-β-lactamase (MβL) activity. The isolates were recovered from different patients in a medical center located in Dusseldorf, Germany. The resistant determinant was isolated amplifying the region between the integrase and the
aacA4
gene cassette. Sequencing revealed a novel MβL gene, designated
bla
GIM-1
. Additional analysis showed that GIM-1, comprising 250 amino acids and with a pI value of 5.4, differs in its primary sequence from that described for IMP, VIM, and SPM-1 enzymes by 39 to 43%, 28 to 31%, and 28%, respectively. The enzyme possesses unique amino acids within the major consensus sequence (HXHXD) of the MβL family. Kinetics analysis revealed that GIM-1 has no clear preference for any substrate and did not hydrolyze azlocillin, aztreonam, and the serine-β-lactamase inhibitors.
bla
GIM-1
was found on a 22-kb nontransferable plasmid. The new MβL gene was embedded in the first position of a 6-kb class 1 integron, In77, with distinct features, including an
aacA4
cassette downstream of the MβL gene that appeared to be truncated with
bla
GIM-1
. The
aacA4
was followed by an
aadA1
gene cassette that was interrupted by a copy of the IS1394. This integron also carried an oxacillinase gene,
bla
OXA-2
, before the 3′-CS region. GIM-1 appears to be a unique MβL, which is located in a distinct integron structure, and represents the fourth subclass of mobile MβL enzymes to be characterized.
Antibacterial Activity of a Competence-Stimulating Peptide in Experimental Sepsis Caused by
Streptococcus pneumoniae
Authors: Oggioni Marco R. , Iannelli Francesco , Ricci Susanna , Chiavolini Damiana , Parigi Riccardo , Trappetti Claudia , Claverys Jean-Pierre , Pozzi Gianni 
Published: November 2004

ABSTRACT

Streptococcus pneumoniae
, a major cause of human disease, produces a 17-mer autoinducer peptide pheromone (competence-stimulating peptide [CSP]) for the control of competence for genetic transformation. Due to previous work linking CSP to stress phenotypes, we set up an in vivo sepsis model to assay its effect on virulence. Our data demonstrate a significant increase in the rates of survival of mice, reductions of blood
S. pneumoniae
counts, and prolonged times to death for mice treated with CSP. In vitro the dose of CSP used in the animal model produced a transitory inhibition of growth. When a mutant with a mutation in the CSP sensor histidine kinase was assayed, no bacteriostatic phenotype was detected in vitro and no change in disease outcome was observed in vivo. The data demonstrate that CSP, which induces in vitro a temporary growth arrest through stimulation of its cognate histidine kinase receptor, is able to block systemic disease in mice. This therapeutic effect is novel, in that the drug-like effect is obtained by stimulation, rather than inhibition, of a bacterial drug target.
Combination of a Hepatitis C Virus NS3-NS4A Protease Inhibitor and Alpha Interferon Synergistically Inhibits Viral RNA Replication and Facilitates Viral RNA Clearance in Replicon Cells
Authors: Lin Kai , Kwong Ann D. , Lin Chao 
Published: November 2004

ABSTRACT
The present standard of care for hepatitis C virus (HCV) infection is pegylated alpha interferon (IFN-α) in combination with ribavirin. However, specific antivirals such as HCV NS3-NS4A protease inhibitors are now in clinical development, and these agents can potentially be used in combination with the present treatments. Therefore, it is important to investigate the potential benefits or adverse effects of these new combinations by using available in vitro HCV culture systems first. In the present study we demonstrate that the combination of a specific HCV NS3-NS4A protease inhibitor and IFN-α synergistically inhibits HCV RNA replication in replicon cells, with little or no increase in cytotoxicity. Furthermore, the benefit of the combination was sustained over time, such that a greater than 3-log reduction in HCV RNA levels was achieved following 9 days of treatment. The viral RNA appeared to be cleared from the replicon cells after 14 days of treatment, and no viral RNA rebound was observed upon withdrawal of the inhibitors. In each case, the antiviral effects obtained with higher concentrations of either the protease inhibitor alone or IFN-α alone can be achieved by a combination of both agents at lower concentrations, which may potentially reduce the risk of possible adverse effects associated with high doses of either agent.
Outbreak of
Klebsiella pneumoniae
Producing a New Carbapenem-Hydrolyzing Class A β-Lactamase, KPC-3, in a New York Medical Center
Authors: Woodford Neil , Tierno Philip M. , Young Katherine , Tysall Luke , Palepou Marie-France I. , Ward Elaina , Painter Ronald E. , Suber Deborah F. , Shungu Daniel , Silver Lynn L. , Inglima Kenneth , Kornblum John , Livermore David M. 
Published: November 2004

ABSTRACT

From April 2000 to April 2001, 24 patients in intensive care units at Tisch Hospital, New York, N.Y., were infected or colonized by carbapenem-resistant
Klebsiella pneumoniae
. Pulsed-field gel electrophoresis identified a predominant outbreak strain, but other resistant strains were also recovered. Three representatives of the outbreak strain from separate patients were studied in detail. All were resistant or had reduced susceptibility to imipenem, meropenem, ceftazidime, piperacillin-tazobactam, and gentamicin but remained fully susceptible to tetracycline. PCR amplified a
bla
KPC
allele encoding a novel variant, KPC-3, with a His(272)→Tyr substitution not found in KPC-2; other carbapenemase genes were absent. In the outbreak strain, KPC-3 was encoded by a 75-kb plasmid, which was transferred in vitro by electroporation and conjugation. The isolates lacked the OmpK35 porin but expressed OmpK36, implying reduced permeability as a cofactor in resistance. This is the third KPC carbapenem-hydrolyzing β-lactamase variant to have been reported in members of the
Enterobacteriaceae
, with others reported from the East Coast of the United States. Although producers of these enzymes remain rare, the progress of this enzyme group merits monitoring.
Two Different Molecular Defects in the Tva Receptor Gene Explain the Resistance of TwotvarLines of Chickens to Infection by Subgroup A Avian Sarcoma and Leukosis Viruses
Authors: Elleder Daniel , Melder Deborah C. , Trejbalova Katerina , Svoboda Jan , Federspiel Mark J. 
Published: November 2004

ABSTRACTThe subgroup A to E avian sarcoma and leukosis viruses (ASLVs) are highly related and are thought to have evolved from a common ancestor. These viruses use distinct cell surface proteins as receptors to gain entry into avian cells. Chickens have evolved resistance to infection by the ASLVs. We have identified the mutations responsible for the block to virus entry in chicken lines resistant to infection by subgroup A ASLVs [ASLV(A)]. Thetvagenetic locus determines the susceptibility of chicken cells to ASLV(A) viruses. In quail, the ASLV(A) susceptibility alleletvasencodes two forms of the Tva receptor; these proteins are translated from alternatively spliced mRNAs. The normal cellular function of the Tva receptor is unknown; however, the extracellular domain contains a 40-amino-acid, cysteine-rich region that is homologous to the ligand binding region of the low-density lipoprotein receptor (LDLR) proteins. The chickentvascDNAs had not yet been fully characterized; we cloned the chickentvacDNAs from two lines of subgroup A-susceptible chickens, line H6 and line 0. Two types of chickentvascDNAs were obtained. These cDNAs encode a longer and shorter form of the Tva receptor homologous to the Tva forms in quail. Two different defects were identified in cDNAs cloned from two different ASLV(A)-resistant inbred chickens, line C and line 72. Line Ctvarcontains a single base pair substitution, resulting in a cysteine-to-tryptophan change in the LDLR-like region of Tva. This mutation drastically reduces the binding affinity of TvaRfor the ASLV(A) envelope glycoproteins. Line 72tvar2contains a 4-bp insertion in exon 1 that causes a change in the reading frame, which blocks expression of the Tva receptor.
Nucleolar Localization of Human Hepatitis B Virus Capsid Protein
Authors: Ning Bo , Shih Chiaho 
Published: November 2004

ABSTRACT
Wild-type human hepatitis B virus (HBV) exhibits selective export of virions containing mature genomes. In contrast, changing an isoleucine to a leucine at amino acid 97 (I97L) of the HBV core antigen (HBcAg) causes it to release immature genomes. To elucidate the structure-function relationship of HBcAg at amino acid 97, we systematically replaced the isoleucine residue at this position with 18 other amino acids via mutagenesis. Twelve of the 18 mutants exhibited no significant phenotype, while five new mutants displayed strong phenotypes. The I97D mutant had a near lethal phenotype, the I97P mutant exhibited a significantly reduced level of virion secretion, and the I97G mutant lacked the full-length relaxed circular form of viral DNA. The tip of the spike of the capsid particle is known to contain a predominant B-cell epitope. However, the recognition of this exposed epitope by an anti-HBc antibody appeared to be affected by the I97E mutation or by histidine tagging at the C terminus of mutant HBcAg, which is presumably in the capsid interior. Surprisingly, the nuclear HBcAg of mutants I97E and I97W, produced from either a replicon or an expression vector, was found to be colocalized with nucleolin and B23 at a frequency of nearly 100% by confocal immunofluorescence microscopy. In contrast, this colocalization occurred with wild-type HBcAg only to a limited extent. We also noted that nucleolin-colocalizing cells were often binucleated or apoptotic, suggesting that the presence of HBcAg in the nucleolus may perturb cytokinesis. The mechanism of this phenomenon and its potential involvement in liver pathogenesis are discussed. To our knowledge, this is the first report of nucleolar HBcAg in culture.
Intracellular Viral Processing, Not Single-Stranded DNA Accumulation, Is Crucial for Recombinant Adeno-Associated Virus Transduction
Authors: Hauck Bernd , Zhao Wei , High Katherine , Xiao Weidong 
Published: November 2004

ABSTRACT
Adeno-associated virus (AAV) is a unique gene transfer vector which takes approximately 4 to 6 weeks to reach its expression plateau. The mechanism for this slow-rise expression profile was proposed to be inefficient second-strand DNA synthesis from the input single-stranded (ss) DNA viral genome. In order to clarify the status of ss AAV genomes, we generated AAV vectors labeled with bromodeoxyuridine (BrdU), a nucleotide analog that can be incorporated into the AAV genome and packaged into infectious virions. Since BrdU-DNA can be detected only by an anti-BrdU antibody when DNA is in an ss form, not in a double-stranded (ds) form, ss AAV genomes with BrdU can be readily tracked in situ. Although ss AAV DNA was abundant by Southern blot analysis, free ss AAV genomes were not detectable after AAV transduction by this new detection method. Further Southern blot analysis of viral DNA and virions revealed that ss AAV DNA was protected within virions. Extracted cellular fractions demonstrated that viral particles in host cells remained infectious. In addition, a significant amount of AAV genomes was degraded after AAV transduction. Therefore, we conclude that the amount of free ss DNA is not abundant during AAV transduction. AAV transduction is limited by the steps that affect AAV ss DNA release (i.e., uncoating) before second-strand DNA synthesis can occur. AAV ss DNA released from viral uncoating is either converted into ds DNA efficiently or degraded by cellular DNA repair mechanisms as damaged DNA. This study elucidates a mechanism that can be exploited to develop new strategies to improve AAV vector transduction efficiency.
Determinants of High Titer in Recombinant Porcine Endogenous Retroviruses
Authors: Harrison Ian , Takeuchi Yasuhiro , Bartosch Birke , Stoye Jonathan P. 
Published: November 2004

ABSTRACT

Porcine endogenous retroviruses (PERVs) pose a potential stumbling block for therapeutic xenotransplantation, with the greatest threat coming from viruses generated by recombination between members of the PERV subgroup A (PERV-A) and PERV-C families (PERV-A/C recombinants). PERV-A and PERV-B have been shown to infect human cells in culture, albeit with low titers. PERV-C has a more restricted host range and cannot infect human cells. A recombinant PERV-A/C virus (PERV-A14/220) contains the PERV-A sequence between the end of
pol
and the middle of the SU region in
env
. The remaining sequence is derived from PERV-C. PERV-A14/220 is approximately 500-fold more infectious than PERV-A. To determine the molecular basis for the increased infectivity of PERV-A14/220, we have made a series of vector constructs. The primary determinant for the enhanced replicative potential of the recombinant virus appeared to be the
env
gene. Using a series of chimeric
env
genes, we could identify two determinants of high infectivity; one was an isoleucine to valine substitution at position 140 between variable regions A and B, and the other lies within the proline rich region. Taken together, these results show that the novel juxtaposition of
env
gene sequences enhanced the infectivity of PERV-A14/220 for human cells, perhaps by stabilization of the envelope glycoprotein or increased receptor binding.
Comparison of Human Immunodeficiency Virus (HIV)-Specific T-Cell Responses in HIV-1- and HIV-2-Infected Individuals in Senegal
Authors: Zheng N. N. , Kiviat N. B. , Sow P. S. , Hawes S. E. , Wilson A. , Diallo-Agne H. , Critchlow C. W. , Gottlieb G. S. , Musey L. , McElrath M. J. 
Published: November 2004

ABSTRACT

Human immunodeficiency virus type 2 (HIV-2) infection is typically less virulent than HIV-1 infection, which may permit the host to mount more effective, sustained T-cell immunity. We investigated antiviral gamma interferon-secreting T-cell responses by an ex vivo Elispot assay in 68 HIV-1- and 55 HIV-2-infected Senegalese patients to determine if differences relate to more efficient HIV-2 control. Homologous HIV-specific T cells were detected in similar frequencies (79% versus 76%,
P
= 0.7) and magnitude (3.12 versus 3.08 log
10
spot-forming cells/10
6
peripheral blood mononuclear cells) in HIV-1 and HIV-2 infection, respectively. Gag-specific responses predominated in both groups (≥64%), and significantly higher Nef-specific responses occurred in HIV-1-infected (54%) than HIV-2-infected patients (22%) (
P
< 0.001). Heterologous responses were more frequent in HIV-1 than in HIV-2 infection (46% versus 27%,
P
= 0.04), but the mean magnitude was similar. Total frequencies of HIV-specific responses in both groups did not correlate with plasma viral load and CD4
+
T-cell count in multivariate regression analyses. However, the magnitude of HIV-2 Gag-specific responses was significantly associated with lower plasma viremia in HIV-1-infected patients (
P
= 0.04). CD4
+
T-helper responses, primarily recognizing HIV-2 Gag, were detected in 48% of HIV-2-infected compared to only 8% of HIV-1-infected patients. These findings indicate that improved control of HIV-2 infection may relate to the contribution of T-helper cell responses. By contrast, the superior control of HIV-1 replication associated with HIV-2 Gag responses suggests that these may represent cross-reactive, higher-avidity T cells targeting epitopes within Gag regions of functional importance in HIV replication.
A DNAβ Associated with
Tomato Yellow Leaf Curl China Virus
Is Required for Symptom Induction
Authors: Cui Xiaofeng , Tao Xiaorong , Xie Yan , Fauquet Claude M. , Zhou Xueping 
Published: November 2004

ABSTRACT

We report here that all 25 isolates of
Tomato yellow leaf curl China virus
(TYLCCNV) collected from tobacco, tomato, or
Siegesbeckia orientalis
plants in different regions of Yunnan Province, China, were associated with DNAβ molecules. To investigate the biological role of DNAβ, full-length infectious clones of viral DNA and DNAβ of TYLCCNV isolate Y10 (TYLCCNV-Y10) were agroinoculated into
Nicotiana benthamiana
,
Nicotiana glutinosa
,
Nicotiana. tabacum
Samsun (NN or nn), tomato, and petunia plants. We found that TYLCCNV-Y10 alone could systemically infect these plants, but no symptoms were induced. TYLCCNV-Y10 DNAβ was required, in addition to TYLCCNV-Y10, for induction of leaf curl disease in these hosts. Similar to TYLCCNV-Y10, DNAβ of TYLCCNV isolate Y64 was also found to be required for induction of typical leaf curl diseases in the hosts tested. When the
βC1
gene of TYLCCNV-Y10 DNAβ was mutated, the mutants failed to induce leaf curl symptoms in
N. benthamiana
when coinoculated with TYLCCNV-Y10. However, Southern blot hybridization analyses showed that the mutated DNAβ molecules were replicated. When
N. benthamiana
and
N. tabacum
plants were transformed with a construct containing the
βC1
gene under the control of the
Cauliflower mosaic virus
35S promoter, many transgenic plants developed leaf curl symptoms similar to those caused by a virus, the severity of which paralleled the level of
βC1
transcripts, while transgenic plants transformed with the
βC1
gene containing a stop codon after the start codon remained symptomless. Thus, expression of a
βC1
gene is adequate for induction of symptoms of viral infection in the absence of virus.