bluuebunny/crossref_metadata_2025 huggingface dataset:
Artemisinin Pharmacokinetics and Efficacy in Uncomplicated-Malaria Patients Treated with Two Different Dosage Regimens
Authors: Gordi Toufigh , Huong Dinh Xuan , Hai Trinh Ngoc , Nieu Nguyen Thi , Ashton Michael
Published: July 2002

The immediate efficacies of two oral dosage regimens of artemisinin were investigated in 77 male and female adult Vietnamese falciparum malaria patients randomly assigned to treatment with either 500 mg of artemisinin daily for 5 days (group A;
n
= 40) or artemisinin at a dose of 100 mg per day for 2 days, with the dose increased to 250 mg per day for 2 consecutive days and with a final dose of 500 mg on the fifth day (group B;
n
= 37). Parasitemia was monitored every 4 h. The average parasite clearance time was longer in group B than in group A (means ± standard deviations, 50 ± 23 and 34 ± 14 h, respectively;
P
< 0.01). Artemisinin concentrations in saliva samples obtained on days 1 and 5 were quantified by high-performance liquid chromatography. The average oral clearance, based on saliva drug concentrations in group B patients, was twofold higher than that in group A patients on day 1 (
P
< 0.01), with no differences in drug half-lives (
P
= 0.40), indicating a saturable first-pass metabolism. Female patients had higher oral clearance values on day 1. Artemisinin's pharmacokinetic parameters were similar on day 5 in both groups, although a significant increase in oral clearance from day 1 to day 5 was evident. Thus, artemisinin exhibited both dose- and time-dependent pharmacokinetics. The escalating dose studied did not result in higher artemisinin concentrations toward the end of the treatment period.
Viral Impacts on Total Abundance and Clonal Composition of the Harmful Bloom-Forming Phytoplankton
Heterosigma akashiwo
Authors: Tarutani Kenji , Nagasaki Keizo , Yamaguchi Mineo
Published: July 2002

Recent observations that viruses are very abundant and biologically active components in marine ecosystems suggest that they probably influence various biogeochemical and ecological processes. In this study, the population dynamics of the harmful bloom-forming phytoplankton
Heterosigma akashiwo
(Raphidophyceae) and the infectious
H. akashiwo
viruses (HaV) were monitored in Hiroshima Bay, Japan, from May to July 1998. Concurrently, a number of
H. akashiwo
and HaV clones were isolated, and their virus susceptibilities and host ranges were determined through laboratory cross-reactivity tests. A sudden decrease in cell density of
H. akashiwo
was accompanied by a drastic increase in the abundance of HaV, suggesting that viruses contributed greatly to the disintegration of the
H. akashiwo
bloom as mortality agents. Despite the large quantity of infectious HaV, however, a significant proportion of
H. akashiwo
cells survived after the bloom disintegration. The viral susceptibility of
H. akashiwo
isolates demonstrated that the majority of these surviving cells were resistant to most of the HaV clones, whereas resistant cells were a minor component during the bloom period. Moreover, these resistant cells were displaced by susceptible cells, presumably due to viral infection. These results demonstrated that the properties of dominant cells within the
H. akashiwo
population change during the period when a bloom is terminated by viral infection, suggesting that viruses also play an important role in determining the clonal composition and maintaining the clonal diversity of
H. akashiwo
populations. Therefore, our data indicate that viral infection influences the total abundance and the clonal composition of one host algal species, suggesting that viruses are an important component in quantitatively and qualitatively controlling phytoplankton populations in natural marine environments.
Composition of Soil Microbial Communities Enriched on a Mixture of Aromatic Hydrocarbons
Authors: Greene E. Anne , Kay Jason G. , Jaber Karim , Stehmeier Les G. , Voordouw Gerrit
Published: July 2002

Soil contaminated with C5+, which contained benzene (45%, wt/wt), dicyclopentadiene (DCPD) plus cyclopentadiene (together 20%), toluene (6%), styrene (3%), xylenes (2%), naphthalene (2%), and smaller quantities of other compounds, served as the source for isolation of 55 genomically distinct bacteria (standards). Use of benzene as a substrate by these bacteria was most widespread (31 of 44 standards tested), followed by toluene (23 of 44), xylenes (14 of 44), styrene (10 of 44), and naphthalene (10 of 44). Master filters containing denatured genomic DNAs of all 55 standards were used to analyze the community compositions of C5+ enrichment cultures by reverse sample genome probing (RSGP). The communities enriched from three contaminated soils were similar to those enriched from three uncontaminated soils from the same site. The compositions of these communities were time dependent and showed a succession of
Pseudomonas
and
Rhodococcus
spp. before convergence on a composition dominated by
Alcaligenes
spp. The dominant community members detected by RSGP were capable of benzene degradation at all stages of succession. The enrichments effectively degraded all C5+ components except DCPD. Overall, degradation of individual C5+ hydrocarbons followed first-order kinetics, with the highest rates of removal for benzene.
Characterization of the Protocatechuic Acid Catabolic Gene Cluster from
Streptomyces
sp. Strain 2065
Authors: Iwagami Sakura G. , Yang Keqian , Davies Julian
Published: July 2002

Protocatechuate 3,4-dioxygenase (EC
1.13.11.3
) catalyzes the ring cleavage step in the catabolism of aromatic compounds through the protocatechuate branch of the β-ketoadipate pathway. A protocatechuate 3,4-dioxygenase was purified from
Streptomyces
sp. strain 2065 grown in
p
-hydroxybenzoate, and the N-terminal sequences of the β- and α-subunits were obtained. PCR amplification was used for the cloning of the corresponding genes, and DNA sequencing of the flanking regions showed that the
pcaGH
genes belonged to a 6.5-kb protocatechuate catabolic gene cluster; at least seven genes in the order
pcaIJFHGBL
appear to be transcribed unidirectionally. Analysis of the cluster revealed the presence of a
pcaL
homologue which encodes a fused γ-carboxymuconolactone decarboxylase/β-ketoadipate enol-lactone hydrolase previously identified in the
pca
gene cluster from
Rhodococcus opacus
1CP. The
pcaIJ
genes encoded proteins with a striking similarity to succinyl-coenzyme A (CoA):3-oxoacid CoA transferases of eukaryotes and contained an indel which is strikingly similar between high-G+C gram-positive bacteria and eukaryotes.
Genetic Diversity among
Arthrobacter
Species Collected across a Heterogeneous Series of Terrestrial Deep-Subsurface Sediments as Determined on the Basis of 16S rRNA and
recA
Gene Sequences
Authors: van Waasbergen Lorraine G. , Balkwill David L. , Crocker Fiona H. , Bjornstad Bruce N. , Miller Robert V.
Published: July 2002

This study was undertaken in an effort to understand how the population structure of bacteria within terrestrial deep-subsurface environments correlates with the physical and chemical structure of their environment. Phylogenetic analysis was performed on strains of
Arthrobacter
that were collected from various depths, which included a number of different sedimentary units from the Yakima Barricade borehole at the U.S. Department of Energy's Hanford site, Washington, in August 1992. At the same time that bacteria were isolated, detailed information on the physical, chemical, and microbiological characteristics of the sediments was collected. Phylogenetic trees were prepared from the 39 deep-subsurface
Arthrobacter
isolates (as well as 17 related type strains) based on 16S rRNA and
recA
gene sequences. Analyses based on each gene independently were in general agreement. These analyses showed that, for all but one of the strata (sedimentary layers characterized by their own unifying lithologic composition), the deep-subsurface isolates from the same stratum are largely monophyletic. Notably, the layers for which this is true were composed of impermeable sediments. This suggests that the populations within each of these strata have remained isolated under constant, uniform conditions, which have selected for a particular dominant genotype in each stratum. Conversely, the few strains isolated from a gravel-rich layer appeared along several lineages. This suggests that the higher-permeability gravel decreases the degree of isolation of this population (through greater groundwater flow), creating fluctuations in environmental conditions or allowing migration, such that a dominant population has not been established. No correlation was seen between the relationship of the strains and any particular chemical or physical characteristics of the sediments. Thus, this work suggests that within sedimentary deep-subsurface environments, permeability of the deposits plays a major role in determining the genetic structure of resident bacterial populations.
Phylogenetic Diversity of Bacteria Associated with the Marine Sponge
Rhopaloeides odorabile
Authors: Webster Nicole S. , Wilson Kate J. , Blackall Linda L. , Hill Russell T.
Published: July 2002

Molecular techniques were employed to document the microbial diversity associated with the marine sponge
Rhopaloeides odorabile
. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the
Actinobacteria
, low-G+C gram-positive bacteria, the β- and γ-subdivisions of the
Proteobacteria
,
Cytophaga/Flavobacterium
, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in
R. odorabile.
High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.
Fluorescent Acid-Fast Microscopy for Measuring Phagocytosis of
Mycobacterium avium
,
Mycobacterium intracellulare
, and
Mycobacterium scrofulaceum
by
Tetrahymena pyriformis
and Their Intracellular Growth
Authors: Strahl Eileen D. , Gillaspy Glenda E. , Falkinham Joseph O.
Published: July 2002

Fluorescent acid-fast microscopy (FAM) was used to enumerate intracellular
Mycobacterium avium
,
Mycobacterium intracellulare
, and
Mycobacterium scrofulaceum
in the ciliated phagocytic protozoan
Tetrahymena pyriformis
. There was a linear relationship between FAM and colony counts of
M. avium
cells both from cultures and within protozoa. The Ziehl-Neelsen acid-fast stain could not be used to enumerate intracellular mycobacteria because uninfected protozoa contained acid-fast, bacterium-like particles. Starved, 7-day-old cultures of
T. pyriformis
transferred into fresh medium readily phagocytized
M. avium
,
M. intracellulare
, and
M. scrofulaceum
. Phagocytosis was rapid and reached a maximum in 30 min.
M. avium
,
M. intracellulare
, and
M. scrofulaceum
grew within
T. pyriformis
, increasing by factors of 4- to 40-fold after 5 days at 30°C. Intracellular
M. avium
numbers remained constant over a 25-day period of growth (by transfer) of
T. pyriformis
. Intracellular
M. avium
cells also survived protozoan encystment and germination. The growth and viability of
T. pyriformis
were not affected by mycobacterial infection. The results suggest that free-living phagocytic protozoa may be natural hosts and reservoirs for
M. avium
,
M. intracellulare
, and
M. scrofulaceum
.
Signature Lipids and Stable Carbon Isotope Analyses of Octopus Spring Hyperthermophilic Communities Compared with Those of
Aquificales
Representatives
Authors: Jahnke Linda L. , Eder Wolfgang , Huber Robert , Hope Janet M. , Hinrichs Kai-Uwe , Hayes John M. , Des Marais David J. , Cady Sherry L. , Summons Roger E.
Published: July 2002

The molecular and isotopic compositions of lipid biomarkers of cultured
Aquificales
genera have been used to study the community and trophic structure of the hyperthermophilic pink streamers and vent biofilm from Octopus Spring.
Thermocrinis ruber
,
Thermocrinis
sp. strain HI 11/12,
Hydrogenobacter
thermophilus
TK-6,
Aquifex pyrophilus
, and
Aquifex aeolicus
all contained glycerol-ether phospholipids as well as acyl glycerides. The
n
-C
20:1
and
cy
-C
21
fatty acids dominated all of the
Aquificales
, while the alkyl glycerol ethers were mainly C
18:0
. These
Aquificales
biomarkers were major constituents of the lipid extracts of two Octopus Spring samples, a biofilm associated with the siliceous vent walls, and the well-known pink streamer community (PSC). Both the biofilm and the PSC contained mono- and dialkyl glycerol ethers in which C
18
and C
20
alkyl groups were prevalent. Phospholipid fatty acids included both the
Aquificales n
-C
20:1
and
cy
-C
21
, plus a series of
iso
-branched fatty acids (
i
-C
15:0
to
i
-C
21:0
), indicating an additional bacterial component. Biomass and lipids from the PSC were depleted in
13
C relative to source water CO
2
by 10.9 and 17.2‰, respectively. The C
20–21
fatty acids of the PSC were less depleted than the
iso
-branched fatty acids, 18.4 and 22.6‰, respectively. The biomass of
T. ruber
grown on CO
2
was depleted in
13
C by only 3.3‰ relative to C source. In contrast, biomass was depleted by 19.7‰ when formate was the C source. Independent of carbon source,
T. ruber
lipids were heavier than biomass (+1.3‰). The depletion in the C
20–21
fatty acids from the PSC indicates that
Thermocrinis
biomass must be similarly depleted and too light to be explained by growth on CO
2
. Accordingly,
Thermocrinis
in the PSC is likely to have utilized formate, presumably generated in the spring source region.
Effect of Solar UV-B Radiation on a Phyllosphere Bacterial Community
Authors: Jacobs Janette L. , Sundin George W.
Published: July 2002

The effect of solar UV-B radiation on the population dynamics and composition of the culturable bacterial community from peanut (
Arachis
hypogeae
L.) was examined in field studies using plants grown under UV-B−transmitting (UV-B+) or UV-B−excluding (UV-B−) plastic filters. Our data demonstrate that solar UV-B selection alters phyllosphere bacterial community composition and that UV tolerance is a prevalent phenotype late in the season. The total bacterial population size was not affected by either UV-B treatment. However, isolates from the UV-B+ plots (
n
= 368) were significantly more UV tolerant than those from the UV-B− (
n
= 363) plots. UV sensitivity was determined as the minimal inhibitory dose of UV that resulted in an inhibition of growth compared to the growth of a nonirradiated control. The difference in minimal inhibitory doses among bacterial isolates from UV-B+ and UV-B− treatments was mainly partitioned among nonpigmented isolates, with pigmented isolates as a group being characterized as UV tolerant. A large increase in UV tolerance was observed within isolate groups collected late (89 and 96 days after planting) in the season. Identification of 200 late-season isolates indicated that the predominant UV-tolerant members of this group were
Bacillus
coagulans
,
Clavibacter
michiganensis
, and
Curtobacterium
flaccumfaciens
. We selected
C
.
michiganensis
as a model UV-tolerant epiphyte to study if cell survival on UV-irradiated peanut leaves was increased relative to UV survival in vitro. The results showed an enhancement in the survival of
C
.
michiganensis
G7.1, especially following high UV-C doses (300 and 375 J m
−2
), that was evident between 24 and 96 h after inoculation. A dramatic increase in the in planta/in vitro survival ratio was observed over the entire 96-h experiment period for
C
.
michiganensis
T5.1.
Differential Synthesis of Peritoxins and Precursors by Pathogenic Strains of the Fungus
Periconia circinata
Authors: Churchill Alice C. L. , Dunkle Larry D. , Silbert Walter , Kennedy Kevin J. , Macko Vlado
Published: July 2002

Pathogenic strains of the soilborne fungus
Periconia circinata
produce peritoxins with host-selective toxicity against susceptible genotypes of sorghum. The peritoxins are low-molecular-weight, hybrid molecules consisting of a peptide and a chlorinated polyketide. Culture fluids from pathogenic, toxin-producing (Tox
+
) and nonpathogenic, non-toxin-producing (Tox
−
) strains were analyzed directly by gradient high-performance liquid chromatography (HPLC) with photodiode array detection and HPLC-mass spectrometry to detect intermediates and final products of the biosynthetic pathway. This approach allowed us to compare the metabolite profiles of Tox
+
and Tox
−
strains. Peritoxins A and B and the biologically inactive intermediates,
N
-3-(
E
-pentenyl)-glutaroyl-aspartate, circinatin, and 7-chlorocircinatin, were detected only in culture fluids of the Tox
+
strains. The latter two compounds were produced consistently by Tox
+
strains regardless of the amount of peritoxins produced under various culture conditions. In summary, none of the known peritoxin-related metabolites were detected in Tox
−
strains, which suggests that these strains may lack one or more functional genes required for peritoxin biosynthesis.
Production of Capsular Polysaccharide of
Streptococcus pneumoniae
Type 14 and Its Purification by Affinity Chromatography
Authors: Suárez Norma , Fraguas Laura Franco , Texeira Esther , Massaldi Hugo , Batista-Viera Francisco , Ferreira Fernando
Published: July 2002

We describe a rapid and efficient method for producing the capsular polysaccharide of
Streptococcus pneumoniae
by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents.
Rapid Method of Determining Factors Limiting Bacterial Growth in Soil
Authors: Aldén L. , Demoling F. , Bååth E.
Published: July 2002

A technique to determine which nutrients limit bacterial growth in soil was developed. The method was based on measuring the thymidine incorporation rate of bacteria after the addition of C, N, and P in different combinations to soil samples. First, the thymidine incorporation method was tested in two different soils: an agricultural soil and a forest humus soil. Carbon (as glucose) was found to be the limiting substance for bacterial growth in both of these soils. The effect of adding different amounts of nutrients was studied, and tests were performed to determine whether the additions affected the soil pH and subsequent bacterial activity. The incubation time required to detect bacterial growth after adding substrate to the soil was also evaluated. Second, the method was used in experiments in which three different size fractions of straw (1 to 2, 0.25 to 1, and <0.25 mm) were mixed into the agricultural soil in order to induce N limitation for bacterial growth. When the straw fraction was small enough (<0.25 mm), N became the limiting nutrient for bacterial growth after about 3 weeks. After the addition of the larger straw fractions (1 to 2 and 0.25 to 1 mm), the soil bacteria were C limited throughout the incubation period (10 weeks), although an increase in the thymidine incorporation rate after the addition of C and N together compared with adding them separately was seen in the sample containing the size fraction from 0.25 to 1 mm. Third, soils from high-pH, limestone-rich areas were examined. P limitation was observed in one of these soils, while tendencies toward P limitation were seen in some of the other soils.
Yeast Population Dynamics during the Fermentation and Biological Aging of Sherry Wines
Authors: Esteve-Zarzoso B. , Peris-Torán M. J. , Garcı́a-Maiquez E. , Uruburu F. , Querol A.
Published: July 2002

Molecular and physiological analyses were used to study the evolution of the yeast population, from alcoholic fermentation to biological aging in the process of “fino” sherry wine making. The four races of “flor”Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis, androuxii) exhibited identical restriction patterns for the region spanning the internal transcribed spacers 1 and 2 (ITS-1 and ITS-2) and the 5.8S rRNA gene, but this pattern was different, from those exhibited by non-florS. cerevisiaestrains. This flor-specific pattern was detected only after wines were fortified, never during alcoholic fermentation, and all the strains isolated from the velum exhibited the typical flor yeast pattern. By restriction fragment length polymorphism of mitochondrial DNA and karyotyping, we showed that (i) the native strain is better adapted to fermentation conditions than commercial strains; (ii) two different populations ofS. cerevisiaestrains are involved in the process of elaboration, of fino sherry wine, one of which is responsible for must fermentation and the other, for wine aging; and (iii) one strain was dominant in the flor population integrating the velum from sherry wines produced in González Byass wineries, although other authors have described a succession of races of florS. cerevisiaeduring wine aging. Analyzing all these results together, we conclude that yeast population dynamics during biological aging is a complex phenomenon and differences between yeast populations from different wineries can be observed.
Dissimilatory Nitrite Reductase Genes from Autotrophic Ammonia-Oxidizing Bacteria
Authors: Casciotti Karen L. , Ward Bess B.
Published: July 2002

The presence of a copper-containing dissimilatory nitrite reductase gene (
nirK
) was discovered in several isolates of β-subdivision ammonia-oxidizing bacteria using PCR and DNA sequencing. PCR primers Cunir3 and Cunir4 were designed based on published
nirK
sequences from denitrifying bacteria and used to amplify a 540-bp fragment of the
nirK
gene from
Nitrosomonas marina
and five additional isolates of ammonia-oxidizing bacteria. Amplification products of the expected size were cloned and sequenced. Alignment of the nucleic acid and deduced amino acid (AA) sequences shows significant similarity (62 to 75% DNA, 58 to 76% AA) between nitrite reductases present in these nitrifiers and the copper-containing nitrite reductase found in classic heterotrophic denitrifiers. While the presence of a nitrite reductase in
Nitrosomonas europaea
is known from early biochemical work, preliminary sequence data from its genome indicate a rather low similarity to the denitrifier
nirK
s. Phylogenetic analysis of the partial nitrifier
nirK
sequences indicates that the topology of the
nirK
tree corresponds to the 16S rRNA and
amoA
trees. While the role of nitrite reduction in the metabolism of nitrifying bacteria is still uncertain, these data show that the
nirK
gene is present in closely related nitrifying isolates from many oceanographic regions and suggest that
nirK
sequences retrieved from the environment may include sequences from ammonia-oxidizing bacteria.
Microarray Analysis of Microbial Virulence Factors
Authors: Chizhikov Vladimir , Rasooly Avraham , Chumakov Konstantin , Levy Dan D.
Published: July 2002

Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of
Escherichia coli
and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (
eaeA
,
slt-I
,
slt-II
,
fliC
,
rfbE
, and
ipaH
) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15
Salmonella
,
Shigella
, and
E. coli
strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.
Effects of Pressure-Induced Membrane Phase Transitions on Inactivation of HorA, an ATP-Dependent Multidrug Resistance Transporter, in
Lactobacillus plantarum
Authors: Ulmer H. M. , Herberhold H. , Fahsel S. , Gänzle M. G. , Winter R. , Vogel R. F.
Published: July 2002

The effects of pressure on cultures of
Lactobacillus plantarum
were characterized by determination of the viability and activity of HorA, an ATP-binding cassette multidrug resistance transporter. Changes in the membrane composition of
L. plantarum
induced by different growth temperatures were determined. Furthermore, the effect of the growth temperature of a culture on pressure inactivation at 200 MPa was determined. Cells were characterized by plate counts on selective and nonselective agar after pressure treatment, and HorA activity was measured by ethidium bromide efflux. Fourier transform-infrared spectroscopy and Laurdan fluorescence spectroscopy provided information about the thermodynamic phase state of the cytoplasmic membrane during pressure treatment. A pressure-temperature diagram for cell membranes was established. Cells grown at 37°C and pressure treated at 15°C lost >99% of HorA activity and viable cell counts within 36 and 120 min, respectively. The membranes of these cells were in the gel phase region at ambient pressure. In contrast, cells grown at 15°C and pressure treated at 37°C lost >99% of HorA activity and viable cell counts within 4 and 8 min, respectively. The membranes of these cells were in the liquid crystalline phase region at ambient pressure. The kinetic analysis of inactivation of
L. plantarum
provided further evidence that inactivation of HorA is a crucial step during pressure-induced cell death. Comparison of the biological findings and the membrane state during pressure treatment led to the conclusion that the inactivation of cells and membrane enzymes strongly depends on the thermodynamic properties of the membrane. Pressure treatment of cells with a liquid crystalline membrane at 0.1 MPa resulted in HorA inactivation and cell death more rapid than those of cells with a gel phase membrane at 0.1 MPa.
Novel α-Glucosidase from
Aspergillus nidulans
with Strong Transglycosylation Activity
Authors: Kato Naoki , Suyama Sachie , Shirokane Masao , Kato Masashi , Kobayashi Tetsuo , Tsukagoshi Norihiro
Published: July 2002

Aspergillus nidulans
possessed an α-glucosidase with strong transglycosylation activity. The enzyme, designated α-glucosidase B (AgdB), was purified and characterized. AgdB was a heterodimeric protein comprising 74- and 55-kDa subunits and catalyzed hydrolysis of maltose along with formation of isomaltose and panose. Approximately 50% of maltose was converted to isomaltose, panose, and other minor transglycosylation products by AgdB, even at low maltose concentrations. The
agdB
gene was cloned and sequenced. The gene comprised 3,055 bp, interrupted by three short introns, and encoded a polypeptide of 955 amino acids. The deduced amino acid sequence contained the chemically determined N-terminal and internal amino acid sequences of the 74- and 55-kDa subunits. This implies that AgdB is synthesized as a single polypeptide precursor. AgdB showed low but overall sequence homology to α-glucosidases of glycosyl hydrolase family 31. However, AgdB was phylogenetically distinct from any other α-glucosidases. We propose here that AgdB is a novel α-glucosidase with unusually strong transglycosylation activity.
Identification of Quorum-Quenching
N
-Acyl Homoserine Lactonases from
Bacillus
Species
Authors: Dong Yi-Hu , Gusti Andi R. , Zhang Qiong , Xu Jin-Ling , Zhang Lian-Hui
Published: July 2002

A range of gram-negative bacterial species use
N
-acyl homoserine lactone (AHL) molecules as quorum-sensing signals to regulate different biological functions, including production of virulence factors. AHL is also known as an autoinducer. An autoinducer inactivation gene,
aiiA
, coding for an AHL lactonase, was cloned from a bacterial isolate,
Bacillus
sp. strain 240B1. Here we report identification of more than 20 bacterial isolates capable of enzymatic inactivation of AHLs from different sources. Eight isolates showing strong AHL-inactivating enzyme activity were selected for a preliminary taxonomic analysis. Morphological phenotypes and 16S ribosomal DNA sequence analysis indicated that these isolates probably belong to the species
Bacillus thuringiensis.
Enzymatic analysis with known
Bacillus
strains confirmed that all of the strains of
B. thuringiensis
and the closely related species
B. cereus
and
B. mycoides
tested produced AHL-inactivating enzymes but
B. fusiformis
and
B. sphaericus
strains did not. Nine genes coding for AHL inactivation were cloned either by functional cloning or by a PCR procedure from selected bacterial isolates and strains. Sequence comparison of the gene products and motif analysis showed that the gene products belong to the same family of AHL lactonases.
Analysis of
Streptococcus mutans
Proteins Modulated by Culture under Acidic Conditions
Authors: Wilkins Joanna C. , Homer Karen A. , Beighton David
Published: July 2002

Streptococcus mutans
, a major etiological agent of dental caries, causes demineralization of the tooth tissue due to the formation of acids from dietary carbohydrates. Dominant among the virulence determinants of this organism are aciduricity and acidogenicity, the abilities to grow at low pH and to produce acid, respectively. The mechanisms underlying the ability of
S. mutans
to survive and proliferate at low pH are currently under investigation. In this study we cultured
S. mutans
at pH 5.2 or 7.0 and extracted soluble cellular proteins. These were analyzed using high-resolution two-dimensional gel electrophoresis, and replicate maps of proteins expressed under each of the two conditions were generated. Proteins with modulated expression at low pH, as judged by a change in the relative integrated optical density, were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption ionization mass spectrometry to generate peptide mass fingerprints, and these were used to assign putative functions according to their homology with the translated sequences in the
S. mutans
genomic database. Thirty individual proteins exhibited altered expression as a result of culture of
S. mutans
at low pH. Up-regulated proteins (
n
= 18) included neutral endopeptidase, phosphoglucomutase, 60-kDa chaperonin, cell division proteins, enolase, lactate dehydrogenase, fructose bisphosphate aldolase, acetoin reductase, superoxide dismutase, and lactoylglutathione lyase. Proteins down-regulated at pH 5.2 (
n
= 12) included protein translation elongation factors G, Tu, and Ts, DnaK, small-subunit ribosomal protein S1P, large-subunit ribosomal protein L12P, and components of both phosphoenolpyruvate:protein phosphotransferase and multiple sugar binding transport systems. The identification of proteins differentially expressed following growth at low pH provides new information regarding the mechanisms of survival and has identified new target genes for mutagenesis studies to further assess their physiological significance.
Comparative Phenotypic, Molecular, and Virulence Characterization of
Vibrio parahaemolyticus
O3:K6 Isolates
Authors: Yeung P. S. Marie , Hayes Micaela C. , DePaola Angelo , Kaysner Charles A. , Kornstein Laura , Boor Kathryn J.
Published: July 2002

Historically,
Vibrio parahaemolyticus
infections have been characterized by sporadic cases caused by multiple, diverse serotypes. However, since 1996,
V. parahaemolyticus
serotype O3:K6 strains have been associated with several large-scale outbreaks of illness, suggesting the emergence of a “new” group of organisms with enhanced virulence. We have applied three different molecular subtyping techniques to identify an appropriate method for differentiating O3:K6 isolates from other serotypes. Pulsed-field gel electrophoresis (PFGE) following
Not
I digestion differentiated seven closely related subtypes among O3:K6 and related strains, which were distinct from PFGE patterns for non-O3:K6 isolates. Ribotyping and
tdh
sequencing were less discriminatory than PFGE, but further confirmed close genetic relationships among recent O3:K6 isolates. In vitro adherence and cytotoxicity studies with human epithelial cells showed that O3:K6 isolates exhibited statistically higher levels of adherence and cytotoxicity to host cells than non-O3:K6 isolates. Epithelial cell cytotoxicity patterns were determined with a lactate dehydrogenase release assay. At 3 h postinfection, high relative cytotoxicities (>50% maximum lactate dehydrogenase activity) were found among a greater proportion of recently isolated O3:K6 and closely related strains (75%) than among the non-O3:K6 isolates (23%). A statistically significant relationship between adherence and cytotoxicity suggests that the pathogenic potential of some isolates may be associated with increased adherence to epithelial cells. Our findings suggest that enhanced adherence and cytotoxicity may contribute to the apparent unique pathogenic potential of
V. parahaemolyticus
O3:K6 strains.
Casein Utilization by
Streptococcus thermophilus
Results in a Diauxic Growth in Milk
Authors: Letort Catherine , Nardi Michèle , Garault Peggy , Monnet Véronique , Juillard Vincent
Published: July 2002

In milk,
Streptococcus thermophilus
displays two distinct exponential growth phases, separated by a nonexponential one, during which proteinase synthesis was initiated. During the second exponential phase, utilization of caseins as the source of amino acids resulted in a decrease in growth rate, presumably caused by a limiting peptide transport activity.
Lipid Removal from Human Serum Samples
Authors: Castro Arnold R. , Morrill William E. , Pope Victoria
Published: July 2002

The efficacy of lipid removal from human serum samples obtained by using Cleanascite HC, a commercially available product, was compared to that obtained by the standard chloroform method. Separate samples of 21 frozen, banked human serum samples used in the preparation of samples for proficiency testing were treated with either Cleanascite HC or chloroform. The lipid content was measured before and after treatment. The total percentages of lipid removed ranged from 61 to 70% with Cleanascite HC and from 60 to 62% with chloroform. The advantage of Cleanascite HC over chloroform is based on the simplicity of the procedure with Cleanascite HC without the environmental concerns inherent in the use of chloroform. In 15 serum samples known to contain antibodies to treponemal and nontreponemal syphilis antigens, Cleanascite HC bound some immunoglobulin, but with only minimal loss of reactivity in the serologic tests for syphilis. Cleanascite HC is therefore an acceptable alternative to chloroform for lipid reduction in human serum samples.
Molecular Markers in Acute and Chronic Phases of Human Toxoplasmosis: Determination of Immunoglobulin G Avidity by Western Blotting
Authors: Marcolino P. T. , Silva D. A. O. , Leser P. G. , Camargo M. E. , Mineo J. R.
Published: July 2002

We characterized antigenic markers recognized by human serum samples from patients presenting with acute and chronic toxoplasmosis by the determination of immunoglobulin G (IgG) antibody avidity by a Western blot modified technique (avidity immunoblotting) that includes the dissociation of the antigen-antibody interaction with 6 or 8 M urea solutions. Human serum samples from 20 patients presenting with recent infection and from 20 patients with chronic infection were analyzed. It was observed that bands p16, p32, p38, p40, p43, p54, p60, p66, and p97 were more frequently recognized by low-avidity IgG in recent infection and by high-avidity IgG in chronic toxoplasmosis. From these antigenic bands, p38 can be characterized as an optimal antigenic marker of low avidity for recent forms of toxoplasmosis due to a significant decrease of their frequencies (from 80 to 0%) after treatment with 6 M urea solutions. The p30 antigen was not considered a good marker to distinguish acute from chronic infection since corresponding IgG antibodies were determined to have high avidity in both phases of the infection. Thus, the avidity immunoblotting assay proved to be a useful tool for determining antigenic markers of recent and chronic phases of
Toxoplasma gondii
infection.
Molecular Confirmation of Human Immunodeficiency Virus (HIV) Type 2 in HIV-Seropositive Subjects in South India
Authors: Kannangai R. , Ramalingam S. , Prakash K. J. , Abraham O. C. , George R. , Castillo R. C. , Schwartz D. H. , Jesudason M. V. , Sridharan G.
Published: July 2002

Nested PCRs for human immunodeficiency virus type 1 (HIV-1) and HIV-2 were compared with immunoblot test results. Twelve of 13 immunoblot-positive HIV-2 samples were positive by PCR. There were five INNO-LIA (Innogenetics, Zwijnaarde, Belgium) and/or HIVBLOT 2.2 (Genelabs, Singapore) samples that tested positive for dual infection. HIV-1 PCR was positive in all samples, while HIV-2 PCR was positive in two and RIBA (Chiron Corporation, San Diego, Calif.) was positive for HIV-2 in three samples. Thus the prevalence of HIV-2 is accurately estimated by the use of immunoblotting, but that of HIV-1 and -2 dual infection may be overestimated.
Comparison of Specific Serological Assays for Diagnosing Human Herpesvirus 6 Infection after Liver Transplantation
Authors: Yoshikawa Tetsushi , Black Jodi B. , Ihira Masaru , Suzuki Kyoko , Suga Sadao , Iida Keiji , Saito Yumiko , Asonuma Katsuhiro , Tanaka Koichi , Asano Yoshizo
Published: July 2002

Cross-reactivity between human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) antibodies and the reliability of specific serological assays were analyzed for 12 patients with concurrent HHV-6 and HHV-7 antibody responses after transplantation with a liver from a living relative by using an immunofluorescence assay (IFA). A neutralizing antibody titer assay (NT) and an immunoblot assay (IB) designed to detect immunoglobulin M (IgM) antibody to the HHV-6 immunodominant 101-kDa protein were compared in the diagnosis of an active HHV-6 infection. A total of 9 of 12 patients demonstrated concurrent HHV-6 and HHV-7 antibody responses, including increased IgG titers and/or the presence of IgM by IFA, and were thus analyzed for cross-reactive antibody to heterologous virus. The average percentages of residual antibody to HHV-6 and HHV-7 after absorption with HHV-6 antigen were 32.6% (range, 6 to 50%) and 55.6% (range, 35 to 100%), respectively. All 12 patients were subsequently analyzed for HHV-6 antibody by using IB and NT. IB detected IgM antibody to the 101-kDa protein in 75% (9 of 12) of the recipients. A significant rise in the NT antibody titer was detected in the same nine samples. However, HHV-6 DNA was detected by PCR in only five of nine plasma samples collected from recipients with a specific serologic response against HHV-6.
Effect of Immunization with Recombinant OspA on Serologic Tests for Lyme Borreliosis
Authors: Fawcett Paul T. , Rose Carlos D. , Budd Sandra M. , Gibney Kathleen M.
Published: July 2002

This study evaluated the effects of vaccination with OspA on the use of serologic tests as aids in the diagnosis of Lyme borreliosis. Sera from control and OspA-immunized mice and from OspA-immunized human volunteers were tested for serologic reactivity to
Borrelia burgdorferi
. Testing was performed with samples obtained prior to administration of vaccine and at 30 days following administration of an initial and a second dose of OspA vaccine. The assays used to assess serologic reactivity included an in-house-developed enzyme-linked immunosorbent assay (ELISA), an in-house-developed Western blot assay, two commercial Western blot tests, and a commercially available dot blot assay. Data obtained from this study demonstrate that immunization with the OspA vaccine will cause ELISA to yield positive results (as reported previously) for the majority of vaccine recipients. Results obtained from Western blot analysis indicate that vaccination with recombinant OspA induces production of antibodies which bind to several different borrelial proteins. The degree of reactivity detected by Western blotting varied greatly between the three assays used. The in-house assay showed the least reactivity, while one commercial Western blot test actually yielded positive test results for infection with
B. burgdorferi
. The usefulness of all three Western blot assays for the diagnosis of potential infection in a vaccine recipient is severely limited by the extensive reactivity caused by vaccination alone. Antibodies produced in response to OspA vaccination did not significantly affect the performance of the dot blot test; thus, it could provide a reliable means to test for infection with
B. burgdorferi
in OspA vaccine recipients.
Optimization of a Human Papillomavirus-Specific Enzyme-Linked Immunosorbent Assay
Authors: Karem Kevin L. , Poon Alysia C. , Bierl Cynthia , Nisenbaum Rosane , Unger Elizabeth
Published: July 2002

A strategy was developed for the control, standardization, and critical evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of human papillomavirus-specific immunoglobulin G in human sera. Control human sera, polyclonal animal sera, and monoclonal antibodies were used to establish optimal assay parameters, including antigen coating, serum dilutions, and criteria for daily reproducibility, monitoring, and rejection of assays. Three evaluation techniques were used in parallel to define an optimal cutoff absorbance value that yields greater than 93% sensitivity and 98.5% specificity in the assay's ability to discriminate positive and negative control sera. This strategy provides an optimal method by which to determine cutoff absorbance values for ELISA.
Comparison of Immunoglobulin G Subclass Profiles Induced by Measles Virus in Vaccinated and Naturally Infected Individuals
Authors: Isa María Beatríz , Martínez Laura , Giordano Miguel , Passeggi Carlos , de Wolff María Cristina , Nates Silvia
Published: July 2002

A total of 258 human sera positive for measles antibodies were divided into four different groups: group 1 contained 54 sera from children after natural measles infection (immunoglobulin M [IgM] positive, early infection phase), group 2 contained 28 sera from children after measles vaccination (IgM positive, early infection phase), group 3 contained 100 sera from healthy adults (natural long-lasting immunity), and group 4 contained 76 sera from healthy children (postvaccinal long-lasting immunity). In the early phase of infection, the percent distributions of measles virus-specific IgG isotypes were similar between natural and postvaccinal immune responses. IgG1 and IgG4 were the dominant isotypes, with mean levels of detection of 100% (natural infection) and 100% (postvaccinal) for IgG1 and 96% (natural infection) and 92% (postvaccinal) for IgG4. In comparison, the IgG4 geometric mean titer (GMT) in the early phase of natural infection was significantly higher than the IgG4 GMT detected in the postvaccinal immune response (80 versus 13; 95% confidence interval). In the memory phase, IgG2 and IgG3 responses decreased significantly in both natural infection and postvaccinal groups, while IgG1 levels were maintained. In contrast, the IgG4 postvaccinal immune response decreased strongly in the memory phase, whereas IgG4 natural long-lasting immunity remained unchanged (9 versus 86%;P< 0.05). The results obtained suggest that IgG4 isotype could be used in the early phase of infection as a quantitative marker and in long-lasting immunity as a qualitative marker to differentiate between natural and postvaccinal immune responses.
Cellular Immune Responses to Recombinant Antigens in Pregnant Women Chronically Infected with
Toxoplasma gondii
Authors: Fatoohi A. F. , Cozon G. J. N. , Greenland T. , Ferrandiz J. , Bienvenu J. , Picot S. , Peyron F.
Published: July 2002

The parasite
Toxoplasma gondii
can infect most mammals and birds, sometimes causing severe pathology. Primary infection during pregnancy can result in abortion or fetal defects. Host immunity, particularly cellular immunity towards antigenic peptides, can control infection, but an efficient vaccine is not yet available. We have evaluated T-cell responses to a crude soluble toxoplasma antigen (ST-Ag) and to five recombinant peptide antigens of cells in whole-blood cultures from 22 pregnant women with preexisting infections and from 7 pregnant negative controls. Cells from all infected patients but from none of the controls responded specifically to ST-Ag by expressing surface CD25 on culture. Responses to the recombinant antigens showed considerable variation between individuals. rGRA1 elicited a response in 16 of the 22 samples (73%), rSAG1 in 13, rGRA7 in 9, rGRA6-CT in 4, and rGRA6-NT in only 1. Most responding cells were CD4
+
. Cells from infected subjects cultured with ST-Ag all released high levels of gamma interferon (IFN-γ) into the culture supernatant (4,343 ± 2,536 pg/ml). Cells from 12 patients released IFN-γ after culture with rGRA1 (130 ± 98 pg/ml), those from 10 patients released it after culture with rSAG1 (183 ± 128 pg/ml), and those from 4 patients released it after culture with rGRA7 (324 ± 374 pg/ml). Intensity of IFN-γ production in response to the latter two recombinant antigens correlated with responses to ST-Ag (
r
= 0.61 and 0.53, respectively;
P
< 0.01). Interleukin-4 was always absent from supernatants of cells stimulated with toxoplasma antigens. The heterogeneity of human responses to individual recombinant toxoplasma antigens should be considered in the design of potential vaccines.
Salmonella
Pathogenicity Island 1-Independent Induction of Apoptosis in Infected Macrophages by
Salmonella enterica
Serotype Typhimurium
Authors: van der Velden Adrianus W. M. , Lindgren Susanne W. , Worley Micah J. , Heffron Fred
Published: July 2002

The enteric pathogen
Salmonella enterica
serotype Typhimurium induces apoptosis in infected macrophages. This process is rapid, specific, and depends on the type III protein secretion system encoded within
Salmonella
pathogenicity island 1 (SPI1). Here, we demonstrate that serotype Typhimurium can activate programmed macrophage cell death independently of SPI1. SPI1 independent induction of apoptosis in infected macrophages is observed as early as 12 to 13 h postinfection, even in the absence of intracellular bacterial replication. Delayed activation of programmed macrophage cell death is not observed with serotype Typhimurium strains mutated in
ompR
or SPI2. Even though SPI2 mutants have a defect in intracellular proliferation, our results indicate that long-term intracellular survival and growth are not required for delayed macrophage killing per se, since
Salmonella
mutants that are severely defective in intracellular growth still induce delayed apoptosis. Inactivation of genes required for either rapid or delayed induction of apoptosis results in a conditional noncytotoxic phenotype, whereas simultaneous inactivation of genes required for both rapid and delayed induction of apoptosis renders serotype Typhimurium noncytotoxic under all conditions tested. Our hypothesis is that differential activation of programmed macrophage cell death by serotype Typhimurium occurs under discrete physiological conditions at distinct locations within an infected host.