bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ultrastructural and Biochemical Alterations Induced by 22,26-Azasterol, a Δ
24(25)
-Sterol Methyltransferase Inhibitor, on Promastigote and Amastigote Forms of
Leishmania amazonensis
Authors: Rodrigues Juliany C. F. , Attias Márcia , Rodriguez Carlos , Urbina Julio A. , Souza Wanderley de 
Published: July 2002

ABSTRACT

We report on the antiproliferative effects and the ultrastructural and biochemical alterations induced in vitro by 22,26-azasterol, a sterol Δ
24(25)
-methyltransferase (24-SMT) inhibitor, on
Leishmania amazonensis
. When promastigotes and amastigotes were exposed to 100 nM 22,26-azasterol, complete growth arrest and cell lysis ensued after 72 (promastigotes) or 120 (amastigotes) h. Exposure of parasites to this azasterol led to the complete depletion of parasite endogenous sterols (episterol and 5-dehydroepisterol) and their replacement by 24-desalkyl sterols (zymosterol, cholesta-5,7,24-trien-3β-ol, and cholesta-7,24-dien-3β-ol), while 14-methyl-zymosterol and 4,14-dimethyl-zymosterol accumulated as a result of simultaneous incubation of the parasites with 22,26-azasterol and ketoconazole, a known inhibitor of the parasite’s sterol C14-demethylase. These results confirmed that 24-SMT is the primary site of action of the azasterol. Profound changes were also observed in the phospholipid compositions of treated cells, in which a twofold reduction in the content of phosphatidylserine was observed; this was accompanied by a concomitant increase in the content of phosphatidylinositol. Transmission electron microscopy showed that 22,26-azasterol induced marked morphological changes, including mitochondrial swelling, invaginations of the inner mitochondrial membrane, and the appearance of large bodies containing concentric membranes. Other modifications included increases in the numbers of acidocalcisomes, megasomes, and lipid inclusions and the appearance of typical autophagic structures and cell body protrusions toward the flagellar pocket. We conclude that the dramatic alteration of the lipid composition of the parasite’s membranes induced by the drug underlies the ultrastructural alterations that lead to the loss of cell viability and that 24-SMT inhibitors could be useful as selective antileishmanial agents.
Broad-Spectrum Antiherpes Activities of 4-Hydroxyquinoline Carboxamides, a Novel Class of Herpesvirus Polymerase Inhibitors
Authors: Oien Nancee L. , Brideau Roger J. , Hopkins Todd A. , Wieber Janet L. , Knechtel Mary L. , Shelly John A. , Anstadt Robert A. , Wells Peter A. , Poorman Roger A. , Huang Audris , Vaillancourt Vallerie A. , Clayton Terrance L. , Tucker John A. , Wathen Michael W. 
Published: July 2002

ABSTRACT
Through broad screening of the compound library at Pharmacia, a naphthalene carboxamide was identified as a nonnucleoside inhibitor of human cytomegalovirus (HCMV) polymerase. Structure-activity relationship studies demonstrated that a quinoline ring could be substituted for naphthalene, resulting in the discovery of a 4-hydroxyquinoline-3-carboxamide (4-HQC) class of antiviral agents with unique biological properties. In vitro assays with the 4-HQCs have demonstrated potent inhibition of HCMV, herpes simplex virus type 1 (HSV-1), and varicella-zoster virus (VZV) polymerases but no inhibition of human α, δ, and γ polymerases. Antiviral cell culture assays have further confirmed that these compounds are active against HCMV, HSV-1, HSV-2, VZV, and many animal herpesviruses. However, these compounds were not active against several nonherpesviruses representing different DNA and RNA virus families. A strong correlation between the viral DNA polymerase and antiviral activity for this class of compounds supports inhibition of the viral polymerase as the mechanism of antiviral activity. Northern blot analysis of immediate-early and late viral transcripts also pointed to a block in the viral life cycle consistent with inhibition of viral DNA replication. In vitro HCMV polymerase assays indicate that the 4-HQCs are competitive inhibitors of nucleoside binding. However, no cross-resistance could be detected with ganciclovir-resistant HCMV or acyclovir-resistant HSV-1 mutants. The unique, broad-spectrum activities of the 4-HQCs may offer new opportunities for treating many of the diseases caused by herpesviruses.
Emergence of Resistance to Protease Inhibitor Amprenavir in Human Immunodeficiency Virus Type 1-Infected Patients: Selection of Four Alternative Viral Protease Genotypes and Influence of Viral Susceptibility to Coadministered Reverse Transcriptase Nucleoside Inhibitors
Authors: Maguire Michael , Shortino Denise , Klein Astrid , Harris Wendy , Manohitharajah Varsha , Tisdale Margaret , Elston Robert , Yeo Jane , Randall Sharon , Xu Fan , Parker Hayley , May Jackie , Snowden Wendy 
Published: July 2002

ABSTRACT

Previous data have indicated that the development of resistance to amprenavir, an inhibitor of the human immunodeficiency virus type 1 protease, is associated with the substitution of valine for isoleucine at residue 50 (I50V) in the viral protease. We present further findings from retrospective genotypic and phenotypic analyses of plasma samples from protease inhibitor-naïve and nucleoside reverse transcriptase inhibitor (NRTI)-experienced patients who experienced virological failure while participating in a clinical trial where they had been randomized to receive either amprenavir or indinavir in combination with NRTIs. Paired baseline and on-therapy isolates from 31 of 48 (65%) amprenavir-treated patients analyzed demonstrated the selection of protease mutations. These mutations fell into four distinct categories, characterized by the presence of either I50V, I54L/I54M, I84V, or V32I+I47V and often included accessory mutations, commonly M46I/L. The I50V and I84V genotypes displayed the greatest reductions in susceptibility to amprenavir, although each of the amprenavir-selected genotypes conferred little or no cross-resistance to other protease inhibitors. There was a significant association, for both amprenavir and indinavir, between preexisting baseline resistance to NRTIs subsequently received during the study and development of protease mutations (
P
= 0.014 and
P
= 0.031, respectively). Our data provide a comprehensive analysis of the mechanisms by which amprenavir resistance develops during clinical use and present evidence that resistance to concomitant agents in the treatment regimen predisposes to the development of mutations associated with protease inhibitor resistance and treatment failure.
Mutations in
ponA
, the Gene Encoding Penicillin-Binding Protein 1, and a Novel Locus,
penC
, Are Required for High-Level Chromosomally Mediated Penicillin Resistance in
Neisseria gonorrhoeae
Authors: Ropp Patricia A. , Hu Mei , Olesky Melanie , Nicholas Robert A. 
Published: July 2002

ABSTRACT

Chromosomally mediated penicillin resistance in
Neisseria gonorrhoeae
occurs in part through alterations in penicillin-binding proteins (PBPs) and a decrease in outer membrane permeability. However, the genetic and molecular mechanisms of transformation of a penicillin-susceptible strain of
N. gonorrhoeae
to high-level penicillin resistance have not been clearly elucidated. Previous studies suggested that alterations in PBP 1 were involved in high-level penicillin resistance. In this study, we identified a single amino acid mutation in PBP 1 located 40 amino acids N terminal to the active-site serine residue that was present in all chromosomally mediated resistant
N. gonorrhoeae
(CMRNG) strains for which MICs of penicillin were ≥1 μg/ml. PBP 1 harboring this point mutation (PBP 1*) had a three- to fourfold lower rate of acylation (
k
2
/
K'
) than wild-type PBP 1 with a variety of β-lactam antibiotics. Consistent with its involvement in high-level penicillin resistance, replacement of the altered
ponA
gene (
ponA1
) in several CMRNG strains with the wild-type
ponA
gene resulted in a twofold decrease in the MICs of penicillin. Surprisingly, transformation of an intermediate-level penicillin-resistant strain (PR100; FA19
penA4 mtr penB5
) with the
ponA1
gene did not increase the MIC of penicillin for this strain. However, we identified an additional resistance locus, termed
penC
, which was required along with
ponA1
to increase penicillin resistance of PR100 to a high level (MIC = 4 μg/ml). The
penC
locus by itself, when present in PR100, increases the MICs of penicillin and tetracycline twofold each. These data indicate that an additional locus,
penC
, is required along with
ponA1
to achieve high-level penicillin resistance.
Identification of a Series of Tricyclic Natural Products as Potent Broad-Spectrum Inhibitors of Metallo-β-Lactamases
Authors: Payne David J. , Hueso-Rodríguez Juan Antonio , Boyd Helen , Concha Néstor O. , Janson Cheryl A. , Gilpin Martin , Bateson John H. , Cheever Christy , Niconovich Nancy L. , Pearson Stewart , Rittenhouse Stephen , Tew David , Díez Emilio , Pérez Paloma , de la Fuente Jesus , Rees Michael , Rivera-Sagredo Alfonso 
Published: July 2002

ABSTRACT

This work describes the discovery and characterization of a novel series of tricyclic natural product-derived metallo-β-lactamase inhibitors. Natural product screening of the
Bacillus cereus
II enzyme identified an extract from a strain of
Chaetomium funicola
with inhibitory activity against metallo-β-lactamases. SB236050, SB238569, and SB236049 were successfully extracted and purified from this extract. The most active of these compounds was SB238569, which possessed

K
i

values of 79, 17, and 3.4 μM for the
Bacillus cereus
II,
Pseudomonas aeruginosa
IMP-1, and
Bacteroides fragilis
CfiA metallo-β-lactamases, respectively, yet none of the compounds exhibited any inhibitory activity against the
Stenotrophomonas maltophilia
L-1 metallo-β-lactamase (50% inhibitory concentration > 1,000 μM). The lack of activity against angiotensin-converting enzyme and serine β-lactamases demonstrated the selective nature of these compounds. The crystal structure of SB236050 complexed in the active site of CfiA has been obtained to a resolution of 2.5 Å. SB236050 exhibits key polar interactions with Lys184, Asn193, and His162 and a stacking interaction with the indole ring of Trp49 in the flap, which is in the closed conformation over the active site groove. SB236050 and SB238569 also demonstrate good antibacterial synergy with meropenem. Eight micrograms of SB236050 per ml gave rise to an eightfold drop in the MIC of meropenem for two clinical isolates of
B. fragilis
producing CfiA, making these strains sensitive to meropenem (MIC ≤ 4 μg/ml). Consequently, this series of metallo-β-lactamase inhibitors exhibit the most promising antibacterial synergy activity so far observed against organisms producing metallo-β-lactamases.
Transformation of 2,4,6-Trinitrotoluene by Purified Xenobiotic Reductase B from
Pseudomonas fluorescens
I-C
Authors: Pak Jeong W. , Knoke Kyle L. , Noguera Daniel R. , Fox Brian G. , Chambliss Glenn H. 
Published: July 2002

ABSTRACT

The enzymatic transformation of 2,4,6-trinitrotoluene (TNT) by purified XenB, an NADPH-dependent flavoprotein oxidoreductase from
Pseudomonas fluorescens
I-C, was evaluated by using natural abundance and [U-
14
C]TNT preparations. XenB catalyzed the reduction of TNT either by hydride addition to the aromatic ring or by nitro group reduction, with the accumulation of various tautomers of the protonated dihydride-Meisenheimer complex of TNT, 2-hydroxylamino-4,6-dinitrotoluene, and 4-hydroxylamino-2,6-dinitrotoluene. Subsequent reactions of these metabolites were nonenzymatic and resulted in predominant formation of at least three dimers with an anionic
m/z
of 376 as determined by negative-mode electrospray ionization mass spectrometry and the release of ∼0.5 mol of nitrite per mol of TNT consumed. The extents of the initial enzymatic reactions were similar in the presence and in the absence of O
2
, but the dimerization reaction and the release of nitrite were favored under aerobic conditions or under anaerobic conditions in the presence of NADP
+
. Reactions of chemically and enzymatically synthesized and high-pressure liquid chromatography-purified TNT metabolites showed that both a hydroxylamino-dinitrotoluene isomer and a tautomer of the protonated dihydride-Meisenheimer complex of TNT were required precursors for the dimerization and nitrite release reactions. The
m/z
376 dimers also reacted with either dansyl chloride or
N
-1-naphthylethylenediamine HCl, providing evidence for an aryl amine functional group. In combination, the experimental results are consistent with assigning the chemical structures of the
m/z
376 species to various isomers of amino-dimethyl-tetranitrobiphenyl. A mechanism for the formation of these proposed TNT metabolites is presented, and the potential enzymatic and environmental significance of their formation is discussed.
Molecular Characterization of Methanotrophic Isolates from Freshwater Lake Sediment
Authors: Auman Ann J. , Stolyar Sergei , Costello Andria M. , Lidstrom Mary E. 
Published: July 2002

ABSTRACT

Profiles of dissolved O
2
and methane with increasing depth were generated for Lake Washington sediment, which suggested the zone of methane oxidation is limited to the top 0.8 cm of the sediment. Methane oxidation potentials were measured for 0.5-cm layers down to 1.5 cm and found to be relatively constant at 270 to 350 μmol/liter of sediment/h. Approximately 65% of the methane was oxidized to cell material or metabolites, a signature suggestive of type I methanotrophs. Eleven methanotroph strains were isolated from the lake sediment and analyzed. Five of these strains classed as type I, while six were classed as type II strains by 16S rRNA gene sequence analysis. Southern hybridization analysis with oligonucleotide probes detected, on average, one to two copies of
pmoA
and one to three copies of 16S rRNA genes. Only one restriction length polymorphism pattern was shown for
pmoA
genes in each isolate, and in cases where, sequencing was done, the
pmoA
copies were found to be almost identical. PCR primers were developed for
mmoX
which amplified 1.2-kb regions from all six strains that tested positive for cytoplasmic soluble methane mono-oxygenase (sMMO) activity. Phylogenetic analysis of the translated PCR products with published
mmoX
sequences showed that MmoX falls into two distinct clusters, one containing the orthologs from type I strains and another containing the orthologs from type II strains. The presence of sMMO-containing
Methylomonas
strains in a pristine freshwater lake environment suggests that these methanotrophs are more widespread than has been previously thought.
Cloning, Sequencing, and Expression of the Pyruvate Carboxylase Gene in
Lactococcus lactis
subsp.
lactis
C2
Authors: Wang H. , O'Sullivan D. J. , Baldwin K. A. , McKay L. L. 
Published: July 2002

ABSTRACT

A functional
pyc
gene was isolated from
Lactococcus lactis
subsp.
lactis
C2 and was found to complement a Pyc defect in
L. lactis
KB4. The deduced lactococcal Pyc protein was highly homologous to Pyc sequences of other bacteria. The
pyc
gene was also detected in
Lactococcus lactis
subsp.
cremoris
and
L. lactis
subsp.
lactis
bv. diacetylactis strains.
Use of Length Heterogeneity PCR and Fatty Acid Methyl Ester Profiles To Characterize Microbial Communities in Soil
Authors: Ritchie Nancy J. , Schutter Mary E. , Dick Richard P. , Myrold David D. 
Published: July 2002

ABSTRACT

In length heterogeneity PCR (LH-PCR) a fluorescently labeled primer is used to determine the relative amounts of amplified sequences originating from different microorganisms. Labeled fragments are separated by gel electrophoresis and detected by laser-induced fluorescence with an automated gene sequencer. We used LH-PCR to evaluate the composition of the soil microbial community. Four soils, which differed in terms of soil type and/or crop management practice, were studied. Previous data for microbial biomass, nitrogen and carbon contents, and nitrogen mineralization rates suggested that the microbial characteristics of these soils were different. One site received two different treatments: no-till and conventional till perennial ryegrass. The other sites were no-till continuous grass plots at separate locations with different soil types. Community composition was characterized by assessing the natural length heterogeneity in eubacterial sequences amplified from the 5′ domain of the 16S rRNA gene and by determining fatty acid methyl ester (FAME) profiles. We found that LH-PCR results were reproducible. Both methods distinguished the three sites. The most abundant bacterial community members, based on cloned LH-PCR products, were members of the β subclass of the class
Proteobacteria
, the
Cytophaga-Flexibacter-Bacteriodes
group, and the high-G+C-content gram-positive bacterial group. Strong correlations were found between LH-PCR results and FAME results. We found that the LH-PCR method is an efficient, reliable, and highly reproducible method that should be a useful tool in future assessments of microbial community composition.
Products from the Incomplete Metabolism of Pyrene by Polycyclic Aromatic Hydrocarbon-Degrading Bacteria
Authors: Kazunga Chikoma , Aitken Michael D. 
Published: July 2002

ABSTRACT

Pyrene is a regulated pollutant at sites contaminated with polycyclic aromatic hydrocarbons (PAH). It is mineralized by some bacteria but is also transformed to nonmineral products by a variety of other PAH-degrading bacteria. We examined the formation of such products by four bacterial strains and identified and further characterized the most apparently significant of these metabolites.
Pseudomonas stutzeri
strain P16 and
Bacillus cereus
strain P21 transformed pyrene primarily to
cis
-4,5-dihydro-4,5-dihydroxypyrene (PYRdHD), the first intermediate in the known pathway for aerobic bacterial mineralization of pyrene.
Sphingomonas yanoikuyae
strain R1 transformed pyrene to PYRdHD and pyrene-4,5-dione (PYRQ). Both strain R1 and
Pseudomonas saccharophila
strain P15 transform PYRdHD to PYRQ nearly stoichiometrically, suggesting that PYRQ is formed by oxidation of PYRdHD to 4,5-dihydroxypyrene and subsequent autoxidation of this metabolite. A pyrene-mineralizing organism,
Mycobacterium
strain PYR-1, also transforms PYRdHD to PYRQ at high initial concentrations of PYRdHD. However, strain PYR-1 is able to use both PYRdHD and PYRQ as growth substrates. PYRdHD strongly inhibited phenanthrene degradation by strains P15 and R1 but had only a minor effect on strains P16 and P21. At their aqueous saturation concentrations, both PYRdHD and PYRQ severely inhibited benzo[
a
]pyrene mineralization by strains P15 and R1. Collectively, these findings suggest that products derived from pyrene transformation have the potential to accumulate in PAH-contaminated systems and that such products can significantly influence the removal of other PAH. However, these products may be susceptible to subsequent degradation by organisms able to metabolize pyrene more extensively if such organisms are present in the system.
Directed Transfer of Large DNA Fragments between
Streptomyces
Species
Authors: Hu Zhihao , Hopwood David A. , Khosla Chaitan 
Published: July 2002

ABSTRACT

The biosynthesis of complex natural products in bacteria is invariably encoded within large gene clusters. Although this facilitates the cloning of such gene clusters, their heterologous expression in genetically amenable hosts remains a challenging problem, principally due to the difficulties associated with manipulating large DNA fragments. Here we describe a new method for the directed transfer of a gene cluster from one
Streptomyces
species to another. The method takes advantage of
tra
gene-mediated conjugal transfer of chromosomal DNA between actinomycetes. As proof of principle, we demonstrate transfer of the entire ∼22-kb actinorhodin gene cluster, and also the high-frequency cotransfer of two loci that are 150 to 200 kb apart, from
Streptomyces coelicolor
to an engineered derivative of
Streptomyces lividans
.
Dynamic Changes of Intracellular pH in Individual Lactic Acid Bacterium Cells in Response to a Rapid Drop in Extracellular pH
Authors: Siegumfeldt Henrik , Björn Rechinger K. , Jakobsen Mogens 
Published: July 2002

ABSTRACT

We describe the dynamics of changes in the intracellular pH (pH
i
) values of a number of lactic acid bacteria in response to a rapid drop in the extracellular pH (pH
ex
). Strains of
Lactobacillus delbrueckii
subsp.
bulgaricus
,
Streptococcus thermophilus
, and
Lactococcus lactis
were investigated.
Listeria innocua
, a gram-positive, non-lactic acid bacterium, was included for comparison. The method which we used was based on fluorescence ratio imaging of single cells, and it was therefore possible to describe variations in pH
i
within a population. The bacteria were immobilized on a membrane filter, placed in a closed perfusion chamber, and analyzed during a rapid decrease in the pH
ex
from 7.0 to 5.0. Under these conditions, the pH
i
of
L. innocua
remained neutral (between 7 and 8). In contrast, the pH
i
values of all of the strains of lactic acid bacteria investigated decreased to approximately 5.5 as the pH
ex
was decreased. No pronounced differences were observed between cells of the same strain harvested from the exponential and stationary phases. Small differences between species were observed with regard to the initial pH
i
at pH
ex
7.0, while different kinetics of pH
i
regulation were observed in different species and also in different strains of
S. thermophilus
.
Biosynthesis of the Lantibiotic Mersacidin: Organization of a Type B Lantibiotic Gene Cluster
Authors: Altena Karsten , Guder André , Cramer Claudia , Bierbaum Gabriele 
Published: July 2002

ABSTRACT

The biosynthetic gene cluster (12.3 kb) of mersacidin, a lanthionine-containing antimicrobial peptide, is located on the chromosome of the producer,
Bacillus
sp. strain HIL Y-85,54728 in a region that corresponds to 348° on the chromosome of
Bacillus subtilis
168. It consists of 10 open reading frames and contains, in addition to the previously described mersacidin structural gene
mrsA
(G. Bierbaum, H. Brötz, K.-P. Koller, and H.-G. Sahl, FEMS Microbiol. Lett. 127:121–126, 1995), two genes,
mrsM
and
mrsD
, coding for enzymes involved in posttranslational modification of the prepeptide; one gene,
mrsT
, coding for a transporter with an associated protease domain; and three genes,
mrsF
,
mrsG
, and
mrsE
, encoding a group B ABC transporter that could be involved in producer self-protection. Additionally, three regulatory genes are part of the gene cluster, i.e.,
mrsR2
and
mrsK2
, which encode a two-component regulatory system which seems to be necessary for the transcription of the
mrsFGE
operon, and
mrsR1
, which encodes a protein with similarity to response regulators. Transcription of
mrsA
sets in at early stationary phase (between 8 and 16 h of culture).