bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Famciclovir on Herpes Simplex Virus Type 1 Corneal Disease and Establishment of Latency in Rabbits
Authors: Loutsch Jeannette M. , Sainz Bruno , Marquart Mary E. , Zheng Xiaodong , Kesavan Prabakaran , Higaki Shiro , Hill James M. , Tal-Singer Ruth
Published: July 2002

Famciclovir (FCV) is efficacious in the treatment of acute herpes zoster and recurrent genital infections but has not been used to treat ocular herpes simplex virus (HSV) infections. We evaluated the efficacy of orally administered FCV in treating HSV-1 epithelial keratitis and determined its effects on the establishment of latency and subsequent reactivation. Rabbits were inoculated with HSV-1 strain 17 syn+ and treated twice daily with increasing concentrations of FCV (60 to 500 mg/kg of body weight). This resulted in a significant, dose-dependent improvement in keratitis scores, as well as prolonged survival. Regardless of the dose of drug used, all groups exhibited the high rates of spontaneous and induced reactivation characteristic of 17syn+. The efficacy of 250 mg of FCV per kg was also compared to topical treatment with 1% trifluorothymidine (TFT). Although TFT treatment was more effective at reducing eye disease, FCV-treated rabbits had a better survival rate. Real-time quantitative PCR analysis of rabbit trigeminal ganglia (TG) demonstrated that FCV significantly reduced the HSV-1 copy number compared to that after treatment with TFT or the placebo but not in a dose-dependent manner. In summary, oral FCV treatment significantly reduces the severity of corneal lesions, reduces the number of HSV-1 genomes in the TG, improves survival, and therefore may be beneficial in reducing the morbidity of HSV keratitis in the clinic.
Molecular Cloning and Characterization of the
Salmonella enterica
Serovar Paratyphi B
rma
Gene, Which Confers Multiple Drug Resistance in
Escherichia coli
Authors: Yassien Mahmoud. A. , Ewis Hosam E. , Lu Chung-Dar , Abdelal Ahmed T.
Published: July 2002

A genomic library from a strain of
Salmonella enterica
serovar Paratyphi B that exhibits multiple drug resistance (MDR) was constructed in
Escherichia coli
. Two of the recombinant plasmids, pNOR5 and pNOR5, conferred resistance only to fluoroquinolones in
E. coli
, whereas the third, pNCTR4, conferred the MDR phenotype. Sequence and subcloning analysis showed that it is the presence of RecA on the first two plasmids which confers resistance to fluoroquinolones in
E. coli
. A similar analysis established that the MDR phenotype conferred by pNCTR4 is due to a gene,
rma
(resistance to multiple antibiotics), which encodes a 13.5-kDa polypeptide. The derived sequence for Rma exhibits a high degree of similarity to those of a group of MarA-like activators that confer MDR in
E. coli
. A MalE-Rma fusion protein was purified to near homogeneity and was shown to interact with a DNA fragment carrying a MarA operator sequence. Furthermore, overexpression of
rma
in
E. coli
caused changes in the outer membrane protein profile that were similar to those reported for MarA. These results suggest that Rma might act as a transcriptional activator of the
marA
regulon.
In Vivo Activities of Farnesyl Pyrophosphate Synthase Inhibitors against
Leishmania donovani
and
Toxoplasma gondii
Authors: Yardley Vanessa , Khan Anis A. , Martin Michael B. , Slifer Teri R. , Araujo Fausto G. , Moreno Silvia N. J. , Docampo Roberto , Croft Simon L. , Oldfield Eric
Published: July 2002

The in vivo activities of three bisphosphonates were determined against
Leishmania donovani
and
Toxoplasma gondii
. Alendronate was essentially inactive against both parasites. Pamidronate was active against
L. donovani
by intravenous administration. Risedronate had a 50% effective dosage of five 2.6-mg/kg of body weight intraperitoneal doses against
L. donovani-
infected mice but was less effective against
T. gondii
-infected mice.
Potency of Nonnucleoside Reverse Transcriptase Inhibitors (NNRTIs) Used in Combination with Other Human Immunodeficiency Virus NNRTIs, NRTIs, or Protease Inhibitors
Authors: King Robert W. , Klabe Ronald M. , Reid Carol D. , Erickson-Viitanen Susan K.
Published: July 2002

Efavirenz and a series of related quinazolinone nonnucleoside inhibitors of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) were evaluated in a series of two-drug combinations with several nucleoside RT inhibitors (NRTIs), nonnucleoside RT inhibitors (NNRTIs), and protease inhibitors (PIs). These combinations were tested in an established HIV-1 RT enzyme assay and a cell-based yield reduction assay with HIV-1 (replicative form [RF])-infected MT-2 cells. Synergy, additivity, and antagonism were determined in the two different assay systems by the method of Chou and Talalay (T.-C. Chou and P. Talalay, Adv. Enzyme Reg. 22:27-55, 1984). Efavirenz, DPC082, DPC083, DPC961, and DPC963 used in combination with the NRTIs zidovudine and lamivudine acted synergistically to inhibit RT activity in the HIV-1 RT enzyme assay and additively to slightly synergistically to inhibit HIV-1 (RF) replication in the yield reduction assay. The five NNRTIs in combination with the PI nelfinavir acted additively in the yield reduction assay to inhibit HIV-1 replication. Interestingly, efavirenz in combination with a second NNRTI acted additively to inhibit HIV-1 RT function in the enzyme assay, while it acted antagonistically to inhibit HIV-1 (RF) replication in the yield reduction assay. These data suggest that antiretroviral combination regimens containing multiple NNTRIs should be given thorough consideration before being used.
Transcriptional Analysis of the
tutE tutFDGH
Gene Cluster from
Thauera aromatica
Strain T1
Authors: Coschigano Peter W.
Published: July 2002

The denitrifying strain T1, identified as
Thauera aromatica
, is able to grow with toluene serving as its sole carbon source. Previous work identified two genes,
tutD
and
tutE
, that are involved in toluene metabolism. Two small open reading frames,
tutF
and
tutG
, which may also play a role in toluene metabolism, were also identified. The present work examines the transcriptional organization and regulation of these toluene utilization genes. Northern analysis indicates that the four genes are organized into two operons,
tutE
and
tutFDG
, and that both operons are regulated in response to toluene. Primer extension analysis has identified major transcriptional start sites located 177 bp upstream of the
tutE
translational start and 76 bp upstream of the
tutF
translational start. Furthermore, a fifth gene,
tutH
, has been identified immediately downstream of
tutG
. It is transcribed from the same start site as
tutFDG
and is predicted to code for a 286-amino-acid protein with a calculated molecular mass of about 31,800 Da. The TutH protein is predicted to have an ATP/GTP binding domain and is similar to the NorQ/NirQ family of proteins.
Transformation of Amoxapine by
Cunninghamella elegans
Authors: Moody Joanna D. , Zhang Donglu , Heinze Thomas M. , Cerniglia Carl E.
Published: July 2002

We examined
Cunninghamella elegans
to determine its ability to transform amoxapine, a tricyclic antidepressant belonging to the dibenzoxazepine class of drugs. Approximately 57% of the exogenous amoxapine was metabolized to three metabolites that were isolated by high-performance liquid chromatography and were identified by nuclear magnetic resonance and mass spectrometry as 7-hydroxyamoxapine (48%),
N
-formyl-7-hydroxyamoxapine (31%), and
N
-formylamoxapine (21%). 7-Hydroxyamoxapine, a mammalian metabolite with biological activity, now can be produced in milligram quantities for toxicological evaluation.
Changes in Glycolytic Activity of
Lactococcus lactis
Induced by Low Temperature
Authors: Wouters Jeroen A. , Kamphuis Henrike H. , Hugenholtz Jeroen , Kuipers Oscar P. , de Vos Willem M. , Abee Tjakko
Published: July 2002

The effects of low-temperature stress on the glycolytic activity of the lactic acid bacterium
Lactococcus lactis
were studied. The maximal glycolytic activity measured at 30°C increased approximately 2.5-fold following a shift from 30 to 10°C for 4 h in a process that required protein synthesis. Analysis of cold adaptation of strains with genes involved in sugar metabolism disrupted showed that both the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) subunit HPr and catabolite control protein A (CcpA) are involved in the increased acidification at low temperatures. In contrast, a strain with the PTS subunit enzyme I disrupted showed increased acidification similar to that in the wild-type strain. This indicates that the PTS is not involved in this response whereas the regulatory function of 46-seryl phosphorylated HPr [HPr(Ser-P)] probably is involved. Protein analysis showed that the production of both HPr and CcpA was induced severalfold (up to two- to threefold) upon exposure to low temperatures. The
las
operon, which is subject to catabolite activation by the CcpA-HPr(Ser-P) complex, was not induced upon cold shock, and no increased lactate dehydrogenase (LDH) activity was observed. Similarly, the rate-limiting enzyme of the glycolytic pathway under starvation conditions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was not induced upon cold shock. This indicates that a factor other than LDH or GAPDH is rate determining for the increased glycolytic activity upon exposure to low temperatures. Based on their cold induction and involvement in cold adaptation of glycolysis, it is proposed that the CcpA-HPr(Ser-P) control circuit regulates this factor(s) and hence couples catabolite repression and cold shock response in a functional and mechanistic way.
Stable Transformation of the
Xylella fastidiosa
Citrus Variegated Chlorosis Strain with
oriC
Plasmids
Authors: Monteiro Patrı́cia B. , Teixeira Diva C. , Palma Renê R. , Garnier Monique , Bové Joseph-Marie , Renaudin Joël
Published: July 2002

Xylella fastidiosa
is a gram-negative, xylem-limited bacterium affecting economically important crops (e.g., grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain of
X. fastidiosa
is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of
X. fastidiosa
were constructed by combining the chromosomal replication origin (
oriC
) of
X. fastidiosa
with a gene which confers resistance to kanamycin (Kan
r
). In plasmid p16KdAori, the
oriC
fragment comprised the
dnaA
gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori the
oriC
fragment was restricted to the
dnaA
-
dnaN
intergenic region, which contains
dnaA
-box like sequences and AT-rich clusters. In plasmid p16K, no
oriC
sequence was present. In the three constructs, the promoter region of one of the two
X. fastidiosa
rRNA operons was used to drive the transcription of the Kan
r
gene to optimize the expression of kanamycin resistance in
X. fastidiosa
. Five CVC
X. fastidiosa
strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori. Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K. Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination. To our knowledge, this is the first report of stable transformation in
X. fastidiosa
. Integration of
oriC
plasmids into the
X. fastidiosa
chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.
Development and Validation ofCorynebacteriumDNA Microarrays
Authors: Loos Andrea , Glanemann Christoph , Willis Laura B. , O'Brien Xian M. , Lessard Philip A. , Gerstmeir Robert , Guillouet Stéphane , Sinskey Anthony J.
Published: July 2002

We have developed DNA microarray techniques for studyingCorynebacterium glutamicum. A set of 52C. glutamicumgenes encoding enzymes from primary metabolism was amplified by PCR and printed in triplicate onto glass slides. Total RNA was extracted from cells harvested during the exponential-growth and lysine production phases of aC. glutamicumfermentation. Fluorescently labeled cDNAs were prepared by reverse transcription using random hexamer primers and hybridized to the microarrays. To establish a set of benchmark metrics for this technique, we compared the variability between replicate spots on the same slide, between slides hybridized with cDNAs from the same labeling reaction, and between slides hybridized with cDNAs prepared in separate labeling reactions. We found that the results were both robust and statistically reproducible. Spot-to-spot variability was 3.8% between replicate spots on a given slide, 5.0% between spots on separate slides (though hybridized with identical, labeled cDNA), and 8.1% between spots from separate slides hybridized with samples from separate reverse transcription reactions yielding an average spot to spot variability of 7.1% across all conditions. Furthermore, when we examined the changes in gene expression that occurred between the two phases of the fermentation, we found that results for the majority of the genes agreed with observations made using other methods. These procedures will be a valuable addition to the metabolic engineering toolbox for the improvement ofC. glutamicumamino acid-producing strains.
Purification and Characterization of a Psychrophilic, Calcium-Induced, Growth-Phase-Dependent Metalloprotease from the Fish Pathogen
Flavobacterium psychrophilum
Authors: Secades P. , Alvarez B. , Guijarro J. A.
Published: July 2002

Flavobacterium psychrophilum
is a fish pathogen that commonly affects salmonids. This bacterium produced an extracellular protease with an estimated molecular mass of 55 kDa. This enzyme, designated Fpp1 (

F

.

p

sychrophilum

p

rotease
1
), was purified to electrophoretic homogeneity from the culture supernatant by using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, and size exclusion chromatography. On the basis of its biochemical characteristics, Fpp1 can be included in the group of metalloproteases that have an optimum pH for activity of 6.5 and are inhibited by 1,10-phenanthroline, EDTA, or EGTA but not by phenylmethylsulfonyl fluoride. Fpp1 activity was dependent on calcium ions not only for its activity but also for its thermal stability. In addition to calcium, strontium and barium can activate the protein. The enzyme showed typical psychrophilic behavior; it had an activation energy of 5.58 kcal/mol and was more active at temperatures between 25 and 40°C, and its activity decreased rapidly at 45°C. Fpp1 cleaved gelatin, laminin, fibronectin, fibrinogen, collagen type IV, and, to a lesser extent, collagen types I and II. Fpp1 also degraded actin and myosin, basic elements of the fish muscular system. The presence of this enzyme in culture media was specifically dependent on the calcium concentration. Fpp1 production started early in the exponential growth phase and reached a maximum during this period. Addition of calcium during the stationary phase did not induce Fpp1 production at all. Besides calcium and the growth phase, temperature also seems to play a role in production of Fpp1. In this study we found that production of Fpp1 depends on factors such as calcium concentration, growth phase of the culture, and temperature. The combination of these parameters corresponds to the combination in the natural host during outbreaks of disease caused by
F. psychrophilum
. Consequently, we suggest that environmental host factors govern Fpp1 production.
Characterization of a Granulovirus Isolated from
Epinotia aporema
Wals. (Lepidoptera: Tortricidae) Larvae
Authors: Sciocco-Cap Alicia , Parola Alejandro D. , Goldberg Alina V. , Ghiringhelli Pablo D. , Romanowski Vı́ctor
Published: July 2002

A granulovirus (GV) isolated from
Epinotia aporema
(Lepidoptera: Tortricidae)—a major soybean pest—was studied in terms of its main morphological, biochemical, and biological properties. The ovoidal occlusion bodies were 466 by 296 nm in size, and their most prominent protein had an apparent molecular mass of 29 kDa. Its amino-terminal sequence was remarkably homologous to that of the granulins of other GVs. The DNA genome size was estimated to be 120 kbp. The high specificity and pathogenicity of this newly described granulovirus (EpapGV) indicate that it is indeed a good candidate for the biological control of this pest.
Identification and Characterization of Bacteria in a Selenium-Contaminated Hypersaline Evaporation Pond
Authors: de Souza M. P. , Amini A. , Dojka M. A. , Pickering I. J. , Dawson S. C. , Pace N. R. , Terry N.
Published: July 2002

Solar evaporation ponds are commonly used to reduce the volume of seleniferous agricultural drainage water in the San Joaquin Valley, Calif. These hypersaline ponds pose an environmental health hazard because they are heavily contaminated with selenium (Se), mainly in the form of selenate. Se in the ponds may be removed by microbial Se volatilization, a bioremediation process whereby toxic, bioavailable selenate is converted to relatively nontoxic dimethylselenide gas. In order to identify microbes that may be used for Se bioremediation, a 16S ribosomal DNA phylogenetic analysis of an aerobic hypersaline pond in the San Joaquin Valley showed that a previously unaffiliated group of uncultured bacteria (belonging to the order
Cytophagales
) was dominant, followed by a group of cultured γ-
Proteobacteria
which was closely related to
Halomonas
species. Se K-edge X-ray absorption spectroscopy of selenate-treated bacterial isolates showed that they accumulated a mixture of predominantly selenate and a selenomethionine-like species, consistent with the idea that selenate was assimilated via the S assimilation pathway. One of these bacterial isolates (
Halomonas
-like strain MPD-51) was the best candidate for the bioremediation of hypersaline evaporation ponds contaminated with high Se concentrations because it tolerated 2 M selenate and 32.5% NaCl, grew rapidly in media containing selenate, and accumulated and volatilized Se at high rates (1.65 μg of Se g of protein
−1
h
−1
), compared to other cultured bacterial isolates.
Self-Protection against Cell Wall Hydrolysis in
Streptococcus milleri
NMSCC 061 and Analysis of the Millericin B Operon
Authors: Beukes Mervyn , Hastings John W.
Published: July 2002

Streptococcus milleri
NMSCC 061 produces an endopeptidase, millericin B, which hydrolyzes the peptide moiety of susceptible cell wall peptidoglycan. The nucleotide sequence of a 4.9-kb chromosomal region showed three open reading frames (ORFs) and a putative tRNA
Leu
sequence. The three ORFs encode a millericin B preprotein (MilB), a putative immunity protein (MilF), and a putative transporter protein (MilT). The
milB
gene encodes a 277-amino-acid preprotein with an 18-amino-acid signal peptide with a consensus IIGG cleavage motif. The predicted protein encoded by
milT
is homologous to ABC (ATP-binding cassette) transporters of several bacteriocin systems and to proteins implicated in the signal-sequence-independent export of
Escherichia coli
hemolysin A. These similarities strongly suggest that the
milT
gene product is involved in the translocation of millericin B. The gene
milF
encodes a protein of 302 amino acids that shows similarities to the FemA and FemB proteins of
Staphylococcus aureus
, which are involved in the addition of glycine to a pentapeptide peptidoglycan precursor. Comparisons of the cell wall mucopeptide of
S. milleri
NMSCC 061(resistant to lysis by millericin B) and
S. milleri
NMSCC 051(sensitive) showed a single amino acid difference. Serial growth of
S. milleri
NMSCC 051 in a cell wall minimal medium containing an increased concentration of leucine resulted in the in vivo substitution of leucine for threonine in the mucopeptide of the cell wall. A cell wall variant of
S. milleri
NMSCC 051 (sensitive) that contained an amino acid substitution (leucine for threonine) within its peptidoglycan cross bridge showed partial susceptibility to millericin B. The putative tRNA
Leu
sequence located upstream of
milB
may be a cell wall-specific tRNA and could together with the
milF
protein, play a potential role in the addition of leucine to the pentapeptide peptidoglycan precursor and thereby, contributing to self-protection to millericin B in the producer strain.
Ammonia- and Nitrite-Oxidizing Bacterial Communities in a Pilot-Scale Chloraminated Drinking Water Distribution System
Authors: Regan John M. , Harrington Gregory W. , Noguera Daniel R.
Published: July 2002

Nitrification in drinking water distribution systems is a common operational problem for many utilities that use chloramines for secondary disinfection. The diversity of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the distribution systems of a pilot-scale chloraminated drinking water treatment system was characterized using terminal restriction fragment length polymorphism (T-RFLP) analysis and 16S rRNA gene (ribosomal DNA [rDNA]) cloning and sequencing. For ammonia oxidizers, 16S rDNA-targeted T-RFLP indicated the presence of
Nitrosomonas
in each of the distribution systems, with a considerably smaller peak attributable to
Nitrosospira
-like AOB. Sequences of AOB amplification products aligned within the
Nitrosomonas oligotropha
cluster and were closely related to
N. oligotropha
and
Nitrosomonas ureae
. The nitrite-oxidizing communities were comprised primarily of
Nitrospira
, although
Nitrobacter
was detected in some samples. These results suggest a possible selection of AOB related to
N. oligotropha
and
N. ureae
in chloraminated systems and demonstrate the presence of NOB, indicating a biological mechanism for nitrite loss that contributes to a reduction in nitrite-associated chloramine decay.
Differential Expression of Methanogenesis Genes of
Methanothermobacter thermoautotrophicus
(Formerly
Methanobacterium thermoautotrophicum
) in Pure Culture and in Cocultures with Fatty Acid-Oxidizing Syntrophs
Authors: Luo Hong-Wei , Zhang Hui , Suzuki Toshihiko , Hattori Satoshi , Kamagata Yoichi
Published: July 2002

The expression of genes involved in methanogenesis in a thermophilic hydrogen-utilizing methanogen,
Methanothermobacter thermoautotrophicus
strain TM, was investigated both in a pure culture sufficiently supplied with H
2
plus CO
2
and in a coculture with an acetate-oxidizing hydrogen-producing bacterium,
Thermacetogenium phaeum
strain PB, in which hydrogen partial pressure was constantly kept very low (20 to 80 Pa). Northern blot analysis indicated that only the
mcr
gene, which encodes methyl coenzyme M reductase I (MRI), catalyzing the final step of methanogenesis, was expressed in the coculture, whereas
mcr
and
mrt
, which encodes methyl coenzyme M reductase II (MRII), the isofunctional enzyme of MRI, were expressed at the early to late stage of growth in the pure culture. In contrast to these two genes, two isofunctional genes (
mtd
and
mth
) for
N
5
,
N
10
-methylene-tetrahydromethanopterin dehydrogenase, which catalyzes the fourth step of methanogenesis, and two hydrogenase genes (
frh
and
mvh
) were expressed both in a pure culture and in a coculture at the early and late stages of growth. The same expression pattern was observed for
Methanothermobacter thermoautotrophicus
strain ΔH cocultured with a thermophilic butyrate-oxidizing syntroph,
Syntrophothermus lipocalidus
strain TGB-C1. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole proteins of
M. thermoautotrophicus
strain TM obtained from a pure culture and a coculture with the acetate-oxidizing syntroph and subsequent N-terminal amino acid sequence analysis confirmed that MRI and MRII were produced in the pure culture, while only MRI was produced in the coculture. These results indicate that under syntrophic growth conditions, the methanogen preferentially utilizes MRI but not MRII. Considering that hydrogenotrophic methanogens are strictly dependent for growth on hydrogen-producing fermentative microbes in the natural environment and that the hydrogen supply occurs constantly at very low concentrations compared with the supply in pure cultures in the laboratory, the results suggest that MRI is an enzyme primarily functioning in natural methanogenic ecosystems.
Microscale Biosensor for Measurement of Volatile Fatty Acids in Anoxic Environments
Authors: Meyer Rikke Louise , Larsen Lars Hauer , Revsbech Niels Peter
Published: July 2002

A microscale biosensor for acetate, propionate, isobutyrate, and lactate is described. The sensor is based on the bacterial respiration of low-molecular-weight, negatively charged species with a concomitant reduction of NO
3
−
to N
2
O. A culture of denitrifying bacteria deficient in N
2
O reductase was immobilized in front of the tip of an electrochemical N
2
O microsensor. The bacteria were separated from the outside environment by an ion-permeable membrane and supplied with nutrients (except for electron donors) from a medium reservoir behind the N
2
O sensor. The signal of the sensor, which corresponded to the rate of N
2
O production, was proportional to the supply of the electron donor to the bacterial mass. The selectivity for volatile fatty acids compared to other organic compounds was increased by selectively enhancing the transport of negatively charged compounds into the sensor by electrophoretic migration (electrophoretic sensitivity control). The sensor was susceptible to interference from O
2
, N
2
O, NO
2
−
, H
2
S, and NO
3
−
. Interference from NO
3
−
was low and could be quantified and accounted for. The detection limit was equivalent to about 1 μM acetate, and the 90% response time was 30 to 90 s. The response of the sensor was not affected by changes in pH between 5.5 and 9 and was also unaffected by changes in salinity in the range of 2 to 32‰. The functioning of the sensor over a temperature span of 7 to 30°C was investigated. The concentration range for a linear response was increased five times by increasing the temperature from 7 to 19.5°C. The life span of the biosensor varied between 1 and 3 weeks after manufacturing.
Identification of a Salt-Induced Primary Transporter for Glycine Betaine in the Methanogen
Methanosarcina mazei
Gö1
Authors: Roeßler M. , Pflüger K. , Flach H. , Lienard T. , Gottschalk G. , Müller V.
Published: July 2002

The salt adaptation of the methanogenic archaeon
Methanosarcina mazei
Gö1 was studied at the physiological and molecular levels. The freshwater organism
M. mazei
Gö1 was able to adapt to salt concentrations up to 1 M, and the addition of the compatible solute glycine betaine to the growth medium facilitated adaptation to higher salt concentrations. Transport studies with cell suspensions revealed a salt-induced glycine betaine uptake activity in
M. mazei
Gö1, and inhibitor studies argue for a primary transport device. Analysis of the genome of
M. mazei
Gö1 identified a homolog of known primary glycine betaine transporters. This gene cluster was designated Ota (osmoprotectant transporter A). Its sequence and gene organization are very similar to those of the glycine betaine transporter OpuA of
Bacillus subtilis
. Northern blot analysis of
otaC
revealed a salt-dependent transcription of this gene. Ota is the first identified salt-induced transporter for compatible solutes in
Archaea
.
A Gene from
Aspergillus nidulans
with Similarity to
URE2
of
Saccharomyces cerevisiae
Encodes a Glutathione
S
-Transferase Which Contributes to Heavy Metal and Xenobiotic Resistance
Authors: Fraser James A. , Davis Meryl A. , Hynes Michael J.
Published: July 2002

Aspergillus nidulans
is a saprophytic ascomycete that utilizes a wide variety of nitrogen sources. We identified a sequence from
A. nidulans
similar to the glutathione
S
-transferase-like nitrogen regulatory domain of
Saccharomyces cerevisiae
Ure2. Cloning and sequencing of the gene, designated
gstA
, revealed it to be more similar to
URE2
than the
S. cerevisiae
glutathione
S
-transferases. However, creation and analysis of a
gstA
deletion mutant revealed that the gene does not participate in nitrogen metabolite repression. Instead, it encodes a functional theta class glutathione
S
-transferase that is involved in resistance to a variety of xenobiotics and metals and confers susceptibility to the systemic fungicide carboxin. Northern analysis showed that
gstA
transcription is strongly activated upon exposure to 1-chloro-2,4-dinitrobenzene and weakly activated by oxidative stress or growth on galactose as a carbon source. These results suggest that nitrogen metabolite repression in
A. nidulans
does not involve a homolog of the
S. cerevisiae URE2
gene and that the global nitrogen regulatory system differs significantly in these two fungi.
Combined Ribotyping and Random Multiprimer DNA Analysis To Probe the Population Structure of
Listeria monocytogenes
Authors: Mereghetti L. , Lanotte P. , Savoye-Marczuk V. , Marquet-Van Der Mee N. , Audurier A. , Quentin R.
Published: July 2002

To improve our understanding of the genetic links between strains originating from food and strains responsible for human diseases, we studied the genetic diversity and population structure of 130 epidemiologically unrelated
Listeria monocytogenes
strains. Strains were isolated from different sources and ecosystems in which the bacterium is commonly found. We used rRNA gene restriction fragment length polymorphism analysis with two endonucleases and random multiprimer DNA analysis with seven oligonucleotide primers to study multiple genetic features of each strain. We used three clustering methods to identify genetic links between individual strains and to determine the precise genetic structure of the population. The combined results confirmed that
L. monocytogenes
strains can be divided into two major phylogenetic divisions. The method used allowed us to demonstrate that the genetic structure and diversity of the two phylogenetic divisions differ. Division I is the most homogeneous and can easily be divided into subgroups with dissimilarity distances of less than 0.30. Each of these subgroups mainly, or exclusively, contains a single serotype (1/2b, 4b, 3b, or 4a). The serotype 4a lineage appears to form a branch that is highly divergent from the phylogenetic group containing serotypes 1/2b, 4b, and 3b. Division II contains strains of serotypes 1/2a, 1/2c, and 3a. It exhibits more genetic diversity with no peculiar clustering. The fact that division II is more heterogeneous than division I suggests that division II evolved from a common ancestor earlier than division I. A significant association was found between division I and human strains, suggesting that strains from division I are better adapted to human hosts.
Butyric Acid-Induced T-Cell Apoptosis Is Mediated by Caspase-8 and -9 Activation in a Fas-Independent Manner
Authors: Kurita-Ochiai Tomoko , Ochiai Kuniyasu , Fukushima Kazuo
Published: July 2002

Our previous study demonstrated that butyric acid, an extracellular metabolite of periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat cells. In this study, we examined whether CD95 ligand-receptor interaction is involved in butyric acid-induced T-cell apoptosis. Flow cytometry analysis indicated that expression of Fas in Jurkat and T cells from peripheral blood mononuclear cells was not affected by butyric acid treatment. Furthermore, the expression of Fas and FasL protein in Western blotting was not affected by butyric acid treatment. Coincubation with blocking anti-Fas antibodies prevented Fas-induced apoptosis but not butyric acid-induced apoptosis. Anti-FasL antibodies also did not prevent butyric acid-induced apoptosis at any dose examined. Although cytotoxic anti-Fas antibody affected butyric acid-induced apoptosis, a synergistic effect was not seen. Time-dependent activation of caspase-8 and -9 was recognized in butyric acid- as well as Fas-mediated apoptosis. IETD-CHO and LEHD-CHO, specific inhibitors of caspase-8 and -9, respectively, completely blocked Fas-mediated apoptosis and partially prevented butyric acid-induced apoptosis. These results suggest that the Fas-FasL interaction is not involved in butyric acid-induced apoptosis and that caspase-8 and -9-dependent apoptosis plays an important role in butyric acid-induced apoptosis, as well as Fas-induced apoptosis.
Antigenic Importance of the Carboxy-Terminal Beta-Strand of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein
Authors: Wootton Sarah , Koljesar Gabriella , Yang Liuzhan , Yoon Kyoung-Jin , Yoo Dongwan
Published: July 2002

Five domains of antigenic importance were previously mapped on the nucleocapsid protein (N) of the porcine reproductive and respiratory syndrome virus (PRRSV), and a domain comprised of the 11 C-terminal-most amino acids (residues 112 to 123) was shown to be essential for binding of N-specific conformation-dependent monoclonal antibodies (MAbs). In the present study, the importance of individual residues within this C-terminal domain for antigenicity was investigated using eight different mutant constructs of N expressed in HeLa cells. Single amino acid substitutions were introduced into the C-terminal domain of the N protein, and the significance of individual amino acids for MAb reactivity was determined by immunoprecipitation. None of the MAbs tested recognized the mutant with a leucine-to-proline substitution at residue 114 (L114P), while V112P, R113P, R113D, I115P, and R116P reduced MAb binding significantly. Conversely, substitution of amino acids at positions 118 (T118S) and 121 (P121A) had little effect on MAb binding. Secondary-structure predictions indicate that amino acids 111 to 117 form a beta-strand. In view of the fact that replacement of beta-strand-forming amino acids with proline elicited the greatest effect on MAb binding, it appears that secondary structure in the C terminus of the N protein is an important determinant of conformational epitope formation. While the crystal structure of the PRRSV N protein remains to be determined, results from these studies broaden our understanding of the secondary structures that make up the PRRSV N protein and shed some light on how they may relate to function.
Reliability of
Helicobacter pylori
and CagA Serological Assays
Authors: Everhart James E. , Kruszon-Moran Deanna , Perez-Perez Guillermo
Published: July 2002

Background serological assays for
Helicobacter pylori
are commonly used without knowledge of reliability. This information is needed to define the ability of serological tests to determine either new cases of infection or loss of infection in longitudinal studies. We evaluated the reproducibility and the interrelationships of serological test results for
H. pylori
and cytotoxin-associated gene product A (CagA) enzyme-linked immunoassays within a subset of participants in a population-based study. Stored samples from 1,229 participants in the third U.S. National Health and Nutrition Examination Survey were replicate serologically tested for
H. pylori
and CagA. Overall disagreement was 3.4% between duplicate tests for
H. pylori
(or 2.3% if equivocal results were disregarded). Six percent of samples positive on the first test had an immune serum ratio at least 30% lower on repeat testing. The odds ratio for
H. pylori
seropositivity on retesting was 2.8 (95% confidence interval [CI] = 1.8 to 4.5) when CagA serology was positive versus when it was negative. CagA antibody was found among 47.8% of
H. pylori
-equivocal and 7.0% of
H. pylori
-negative samples. CagA-positive yet
H. pylori
-negative samples were more likely to occur among Mexican Americans (odds ratio, 5.2; 95% CI = 2.4 to 11.4) and non-Hispanic blacks (odds ratio, 5.5; 95% CI = 2.3 to 13.0) than among non-Hispanic whites. Relying on repeated
H. pylori
serological tests over time to determine infection rates may result in misinterpretation due to limits in test reproducibility. CagA testing may have a role in verifying infection.
Induction of Protective Immunity againstChlamydia trachomatisGenital Infection by a Vaccine Based on Major Outer Membrane Protein–Lipophilic Immune Response-Stimulating Complexes
Authors: Igietseme Joseph U. , Murdin Andrew
Published: July 2002

The significance of delivery systems in modern vaccine design strategies is underscored by the fact that a promising vaccine formulation may fail in vivo due to an inappropriate delivery method. We evaluated the immunogenicity and efficacy of a candidate vaccine comprising the major outer membrane protein (MOMP) ofChlamydia trachomatisdelivered with the lipophilic immune response-stimulating complexes (ISCOMs) as a vehicle with adjuvant properties, in a murine model of chlamydial genital infection. Immunocompetent BALB/c mice were immunized intranasally (IN) or intramuscularly (IM) with MOMP, MOMP-ISCOMs, and live or heat-inactivatedC. trachomatisserovar D. The level of local genital mucosal Th1 response was measured by assaying for antigen-specific Th1 cell induction and recruitment into the genital mucosa at different times after immunization. Immunization with MOMP-ISCOMs by the IM route induced the greatest and fastest local genital mucosal Th1 response, first detectable 2 weeks after exposure. Among the other routes and regimens tested, only IN immunization with MOMP-ISCOMs induced detectable and statistically significant levels of local genital mucosal Th1 response during the 8-week test period (P< 0.001). In addition, when T cells from immunized mice were adoptively transferred into syngeneic naive animals and challenged intravaginally withChlamydia, recipients of IM immunization of MOMP-ISCOMs cleared their infection within 1 week and were resistant to reinfection. Animals that received IN immunization of MOMP-ISCOMs were partially protected, shedding fewer chlamydiae than did control mice. Altogether, the results suggested that IM delivery of MOMP-ISCOMs may be a suitable vaccine regimen potentially capable of inducing protective mucosal immunity againstC. trachomatisinfection.
Listeria monocytogenesas a Short-Lived Delivery System for the Induction of Type 1 Cell-Mediated Immunity against the p36/LACK Antigen ofLeishmania major
Authors: Soussi Neirouz , Milon Geneviève , Colle Jean-Hervé , Mougneau Evelyne , Glaichenhaus Nicolas , Goossens Pierre L.
Published: July 2002

Listeria monocytogeneshas been used as an experimental live vector for the induction of CD8-mediated immune responses in various viral and tumoral experimental models. Susceptibility of BALB/c mice toLeishmania majorinfection has been correlated to the preferential development of Th2 CD4 T cells through an early production of interleukin 4 (IL-4) by a restricted population of CD4 T cells which react to a single parasite antigen, LACK (stands forLeishmaniahomologue of receptors for activated C kinase). Experimental vaccination with LACK can redirect the differentiation of CD4+T cells towards the Th1 pathway if LACK is coadministrated with IL-12. As IL-12 is known to be induced byL. monocytogenes, we have tested the ability of a recombinant attenuatedactAmutantL. monocytogenesstrain expressing LACK to induce the development of LACK-specific Th1 cells in both B10.D2 and BALB/c mice, which are resistant and susceptible toL. major, respectively. After a single injection of LACK-expressingL. monocytogenes, IL-12/p40 transcripts showed a rapid burst, and peaks of gamma interferon (IFN-γ)-secreting LACK-specific Th1 cells were detected around day 5 in the spleens and livers of mice of both strains. These primed IFN-γ-secreting LACK-reactive T cells were not detected ex vivo after day 7 of immunization but could be recruited and detected 15 days later in the draining lymph node after anL. majorfootpad challenge. Although immunization of BALB/c mice with LACK-expressingL. monocytogenesdid not change the course of the infection withL. major, immunized B10.D2 mice exhibited significantly smaller lesions than nonimmunized controls. Thus, our results demonstrate that, in addition of its recognized use for the induction of effector CD8 T cells,L. monocytogenescan also be used as a live recombinant vector to favor the development of potentially protective IFN-γ-secreting Th1 CD4 T lymphocytes.
Cell Vacuolation, a Manifestation of the El Tor Hemolysin of
Vibrio cholerae
Authors: Mitra Rupak , Figueroa Paula , Mukhopadhyay Asish K. , Shimada Toshio , Takeda Yoshifumi , Berg Douglas E. , Nair G. Balakrish
Published: July 2002

Culture supernatants of nontoxigenic nonepidemic clinical strains of
Vibrio cholerae
belonging to diverse serogroups were found to induce vacuolation of nonconfluent HeLa cells. The vacuoles became prominent 18 h after introduction of culture supernatant, and vacuolated cells survived for 48 h and then died. Only a fraction of the vacuolated cells took up neutral red dye, implying that there were differences in the vacuolar microenvironment. Further tests showed that the factor responsible for vacuolation was heat labile and proteinaceous. Vacuolating activity was completely neutralized by antibody to hemolysin of
V. cholerae
but not by antibody to vacuolating cytotoxin of
Helicobacter pylori
. Partial purification of the vacuolating factor led to elution of fractions, which showed both hemolytic and vacuolating activity. PCR amplification and cloning of the hemolysin structural gene (
hlyA
) into
Escherichia coli
DH5α led to isolation of clones producing cell vacuolating factor in a cell-associated form. Further, a null insertion mutation in the
hlyA
gene of a high-vacuolating-factor-producing strain led to complete abolition of both cell vacuolating and hemolytic activities. These analyses establish vacuolation as a potentially important but previously unrecognized property of
V. cholerae
El Tor hemolysin.
Effects of Shiga Toxin 2 on Lethality, Fetuses, Delivery, and Puerperal Behavior in Pregnant Mice
Authors: Yoshimura Kazuaki , Fujii Jun , Tanimoto Akihide , Yutsudo Takashi , Kashimura Masamichi , Yoshida Shin-ichi
Published: July 2002

Shiga toxin 2 (Stx2) is produced by enterohemorrhagic
Escherichia coli
(EHEC) and is known as the major virulence factor of EHEC. The aim of this study was to evaluate the effects of Stx2 on (i) maternal lethality, (ii) fetuses, (iii) delivery period, and (iv) maternal behavior after delivery. Timed pregnant ICR mice were injected intravenously with Stx2 on day 5 of pregnancy (early stage) or on day 15 (late stage). In early-stage experiments, the number of normal fetuses of mice injected with Stx2 was significantly lower than that of control mice. In late-stage experiments, mothers injected with Stx2 delivered normal numbers of neonates, but could not take care of them. The lethal doses of Stx2 were not different for pregnant and nonpregnant female mice at either stage. We conclude that Stx2 is toxic to the fetus in early pregnancy and affects maternal puerperal behavior in late pregnancy.
Interleukin-4 and Interleukin-10 Are Involved in Host Resistance toStaphylococcus aureusInfection through Regulation of Gamma Interferon
Authors: Sasaki Sanae , Nishikawa Shinsuke , Miura Tomisato , Mizuki Mayuko , Yamada Kyogo , Madarame Hiroo , Tagawa Yoh-Ichi , Iwakura Yoichiro , Nakane Akio
Published: July 2002

Our previous study showed that gamma interferon (IFN-γ), a T-helper 1 (Th1)-type cytokine, plays a detrimental role inStaphylococcus aureusinfection in mice. In this study, the role of Th2-type cytokines such as interleukin-4 (IL-4) and IL-10 inS. aureusinfection was investigated. IL-10 mRNA was induced in parallel with IFN-γ in the spleens and kidneys of mice duringS. aureusinfection, whereas IL-4 mRNA was induced in the spleens but not in the kidneys of these animals. Spleen cells obtained fromS. aureus-infected mice produced lower titers of IFN-γ and higher titers of IL-4 and IL-10 in response to heat-killedS. aureusthan did those from uninfected mice. Administration of anti-IL-4 monoclonal antibody (MAb) or anti-IL-10 MAb inhibited the elimination ofS. aureuscells from the kidneys of mice. IFN-γ mRNA expression was enhanced in the spleens of anti-IL-4 MAb- or anti-IL-10 MAb-treated mice and also in the kidneys of anti-IL-4 MAb-treated animals. Next, we evaluated the role of IFN-γ inS. aureusinfection in IFN-γ−/−mice. An increase in survival rates, a decrease in bacterial numbers in the kidneys, and an amelioration of histologic abnormalities in these organs were observed in IFN-γ−/−mice compared with those in IFN-γ+/+mice. Administration of MAb against IL-4 or IL-10 failed to affect bacterial growth in the spleens and kidneys of IFN-γ−/−mice irrespective of the expression of Th2 response. These results suggest thatS. aureusinfection induced a Th2 response and that IL-4 and IL-10 might play a protective role through the regulation of IFN-γ inS. aureusinfection.
Yersinia enterocoliticaInvasin Protein Triggers Differential Production of Interleukin-1, Interleukin-8, Monocyte Chemoattractant Protein 1, Granulocyte-Macrophage Colony-Stimulating Factor, and Tumor Necrosis Factor Alpha in Epithelial Cells: Implications for Understanding the Early Cytokine Network inYersiniaInfections
Authors: Kampik Daniel , Schulte Ralf , Autenrieth Ingo B.
Published: July 2002

Yersinia enterocoliticainfection of epithelial cells results in interleukin-8 (IL-8) mRNA expression. Herein we demonstrate that besides IL-8, increased mRNA levels of five other cytokines, IL-1α, IL-1β, monocyte chemoattractant protein 1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-α), can be detected upon infection of HeLa cells withYersinia. Yersinia-triggered cytokine production was not affected by blocking phosphatidylinositol-3-phosphate kinase with wortmannin, which inhibited bacterial invasion. Comparable cytokine mRNA responses were triggered byEscherichia coliexpressingYersinia inv, while no response was triggered by aninv-deficientYersiniamutant. Moreover, cytokine responses were independent from metabolic activity of the bacteria, as killed bacterial cells were sufficient for triggering cytokine responses in HeLa cells. Semiquantitative reverse transcription-PCR analysis was used to assess the kinetics of cytokine mRNA expression in infected HeLa cells. IL-8, IL-1α, IL-1β, MCP-1, GM-CSF, and TNF-α mRNA expression increased within 1 h postinfection, reached a maximum after 3 to 4 h, and then declined to preinfection levels within 3 h. IL-8, MCP-1, and GM-CSF were secreted by HeLa cells, whereas IL-1α and IL-1β were not secreted and thus were found exclusively intracellularly. TNF-α protein could not be detected in cell lysates or supernatants. Stimulation of HeLa cells with IL-1α was followed by increased IL-8 mRNA expression, whereas stimulation with IL-8 did not induce cytokine production. Likewise, MCP-1 and GM-CSF did not induce significant cytokine responses in HeLa cells. Our results implicate that the initial host response toYersiniainfection might be sustained by IL-8, MCP-1, and GM-CSF produced by epithelial cells.
Transforming Growth Factor β1 Is Expressed in the Jejunum after ExperimentalCryptosporidium parvumInfection in Humans
Authors: Robinson Prema , Okhuysen Pablo C. , Chappell Cynthia L. , Lewis Dorothy E. , Shahab Imran , Lahoti Sandeep , White A. Clinton
Published: July 2002

Biopsies from volunteers challenged withCryptosporidium parvumwere examined for transforming growth factor β1 (TGF-β1). None of the prechallenge biopsies exhibited TGF-β. Seven of 12 volunteers with oocyst shedding expressed TGF-β versus 2 of 13 volunteers without detected oocysts. The association of TGF-β expression with oocyst excretion and the timing of symptoms suggests that TGF-β mediates intestinal healing.