bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Ca2+ Status of the Endoplasmic Reticulum Is Altered by Induction of Calreticulin Expression in Transgenic Plants
Authors: Persson Staffan , Wyatt Sarah E. , Love John , Thompson William F. , Robertson Dominique , Boss Wendy F. 
Published: July 2002

Abstract
To investigate the endoplasmic reticulum (ER) Ca2+ stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca2+-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca2+uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent 45Ca2+accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca2+ ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of45Ca2+ released, and a 2- to 3-fold increase in the amount of 45Ca2+ retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca2+ pool. In addition, CRTtransgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca2+-containing medium to Ca2+-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca2+ stores and thereby enhances the survival of plants grown in low Ca2+ medium.
Antimalarial Activities of Dermaseptin S4 Derivatives
Authors: Krugliak Miriam , Feder Rina , Zolotarev Vadim Y. , Gaidukov Leonid , Dagan Arie , Ginsburg Hagai , Mor Amram 
Published: July 2002

ABSTRACT

The hemolytic antimicrobial peptide dermaseptin S4 was recently shown to exert antimalarial activity. In this study, we attempted to understand the underlying mechanism(s) and identify derivatives with improved antimalarial activity. A number of dermaseptin S4 derivatives inhibited parasite growth with a 50% inhibitory concentration (IC
50
) in the micromolar range. Among these, the substituted S4 analog K
4
K
20
-S4 was the most potent (IC
50
= 0.2 μM), while its shorter version, K
4
-S4(1–13)a, retained a considerable potency (IC
50
= 6 μM). Both K
4
K
20
-S4 and K
4
-S4(1–13)a inhibited growth of the parasites more at the trophozoite stage than at the ring stage. Significant growth inhibition was observed after as little as 1 min of exposure to peptides and proceeded with nearly linear kinetics. The peptides selectively lysed infected red blood cells (RBC) while having a weaker effect on noninfected RBC. Thus, K
4
K
20
-S4 lysed trophozoites at concentrations similar to those that inhibited their proliferation, but trophozoites were >30-fold more susceptible than normal RBC to the lytic effect of K
4
K
20
-S4, the most hemolytic dermaseptin. The same trend was observed with K
4
-S4(1–13)a. The
d
isomers of K
4
K
20
-S4 or K
4
-S4(1–13)a were as active as the
l
counterparts, indicating that antimalarial activity of these peptides, like their membrane-lytic activity, is not mediated by specific interactions with a chiral center. Moreover, dissipation of transmembrane potential experiments with infected cells indicated that the peptides induce damage in the parasite's plasma membrane. Fluorescence confocal microscopy analysis of treated infected cells also indicated that the peptide is able to find its way through the complex series of membranes and interact directly with the intracellular parasite. Overall, the data showed that dermaseptins exert antimalarial activity by lysis of infected cells. Dermaseptin derivatives are also able to disrupt the parasite plasma membrane without harming that of the host RBC.
Role of Penicillin-Binding Protein 4 in Expression of Vancomycin Resistance among Clinical Isolates of Oxacillin-Resistant
Staphylococcus aureus
Authors: Finan J. E. , Archer Gordon L. , Pucci Michael J. , Climo Michael W. 
Published: July 2002

ABSTRACT

It has been reported that penicillin-binding protein 4 (PBP4) activity decreases when a vancomycin-susceptible
Staphylococcus aureus
isolate is passaged in vitro to vancomycin resistance. We analyzed the PBP profiles of four vancomycin intermediately susceptible
S. aureus
(VISA) clinical isolates and found that PBP4 was undetectable in three isolates (HIP 5827, HIP 5836, and HIP 6297) and markedly reduced in a fourth (Mu50). PBP4 was readily visible in five vancomycin-susceptible, oxacillin-resistant
S. aureus
(ORSA) isolates. The nucleotide sequences of the
pbp4
structural gene and flanking sequences did not different between the VISA and vancomycin-susceptible isolates. Overproduction of PBP4 on a high-copy-number plasmid in the VISA isolates produced a two- to threefold decrease in vancomycin MICs. Inactivation of
pbp4
by allelic replacement mutagenesis in three vancomycin-susceptible ORSA strains (COL, RN450M, and N315) led to a decrease in vancomycin susceptibility, an increase in highly vancomycin-resistant subpopulations, and decreased cell wall cross-linking by high-performance liquid chromatography analysis. Complementation of the COL mutant with plasmid-encoded
pbp4
restored the vancomycin MIC and increased cell wall cross-linking. These data suggest that alterations in PBP4 expression are at least partially responsible for the VISA phenotype.
Cloning and Biochemical Characterization of FOX-5, an AmpC-Type Plasmid-Encoded β-Lactamase from a New York City
Klebsiella pneumoniae
Clinical Isolate
Authors: Queenan Anne Marie , Jenkins Stephen , Bush Karen 
Published: July 2002

ABSTRACT

Klebsiella pneumoniae
5064, isolated in New York, carried plasmid-mediated resistance to multiple β-lactams and was unresponsive to clavulanic acid. The β-lactamase gene responsible for cephalosporin resistance encoded FOX-5, with 96 to 97% amino acid identities to other members of the FOX family of β-lactamases. The
bla
FOX-5
coding region was located next to a transposase gene from the
Aeromonas salmonicida
insertion element IS
AS2
.
In Vivo Monitoring of Intracellular ATP Levels inLeishmania donovaniPromastigotes as a Rapid Method To Screen Drugs Targeting Bioenergetic Metabolism
Authors: Luque-Ortega Juan Román , Rivero-Lezcano Octavio Miguel , Croft Simon L. , Rivas Luis 
Published: July 2002

ABSTRACTA method for the rapid screening of drugs targeting the bioenergetic metabolism ofLeishmaniaspp. was developed. The system is based on the monitoring of changes in the intracellular ATP levels ofLeishmania donovanipromastigotes that occur in vivo, as assessed by the luminescence produced by parasites transfected with a cytoplasmic form ofPhothinus pyralisluciferase and incubated with free-membrane permeabled-luciferin analogued-luciferin–[1-(4,5-dimethoxy-2-nitrophenyl) ethyl ester]. A significant correlation was obtained between the rapid inhibition of luminescence with parasite proliferation and the dissipation of changes in mitochondrial membrane potential (ΔΨm) produced by buparvaquone or plumbagin, two leishmanicidal inhibitors of oxidative phosphorylation. To further validate this test, a screen of 14 standard leishmanicidal drugs, using a 50 μM cutoff, was carried out. Despite its semiquantitative properties and restriction to the promastigote stage, this test compares favorably with other bioenergetic parameters with respect to time and cell number requirements for the screening of drugs that affect mitochondrial activity.
Safety and Pharmacokinetics of Multiple 750-Milligram Doses of Intravenous Levofloxacin in Healthy Volunteers
Authors: Chow Andrew T. , Fowler Cynthia , Williams R. Rex , Morgan Nancy , Kaminski Susan , Natarajan Jaya 
Published: July 2002

ABSTRACT
The safety and pharmacokinetics of a once-daily high intravenous dose of levofloxacin (750 mg) in 18 healthy volunteers were studied in a double-blind, randomized, placebo-controlled, single-center parallel group study. Levofloxacin was well tolerated, and higher maximum concentration of drug in serum and area under the concentration-time curve values were achieved. For difficult-to-treat infections, high daily doses of levofloxacin may be beneficial, and intravenous administration may be preferred in certain clinical settings, such as when treating patients in intensive care units, warranting further evaluation.
Three Cefotaximases, CTX-M-9, CTX-M-13, and CTX-M-14, among
Enterobacteriaceae
in the People's Republic of China
Authors: Chanawong Aroonwadee , M'Zali Fatima Hannachi , Heritage John , Xiong Jian-Hui , Hawkey Peter Michael 
Published: July 2002

ABSTRACT

Of 15 extended-spectrum β-lactamase (ESBL)-producing isolates of the family
Enterobacteriaceae
collected from the First Municipal People's Hospital of Guangzhou, in the southern part of the People's Republic of China, 9 were found to produce CTX-M ESBLs, 3 produced SHV-12, and 3 produced both CTX-M and SHV-12. Eleven isolates produced either TEM-1B or SHV-11, in addition to an ESBL. Nucleotide sequence analysis of the 12 isolates carrying
bla
CTX-M
genes revealed that they harbored three different
bla
CTX-M
genes,
bla
CTX-M-9
(5 isolates),
bla
CTX-M-13
(1 isolate), and
bla
CTX-M-14
(6 isolates). These genes have 98% nucleotide homology with
bla
Toho-2
. The
bla
CTX-M
genes were carried on plasmids that ranged in size from 35 to 150 kb. Plasmid fingerprints and pulsed-field gel electrophoresis showed the dissemination of the
bla
CTX-M
genes through transfer of different antibiotic resistance plasmids to different bacteria, suggesting that these resistance determinants are highly mobile. Insertion sequence IS
Ecp1
, found on the upstream region of these genes, may be involved in the translocation of the
bla
CTX-M
genes. This is the first report of the occurrence of SHV-12 and CTX-M ESBLs in China. The presence of strains with these ESBLs shows both the evolution of
bla
CTX-M
genes and their dissemination among at least three species of the family
Enterobacteriaceae
,
Escherichia coli
,
Klebsiella pneumoniae
, and
Enterobacter cloacae
, isolated within a single hospital. The predominance of CTX-M type enzymes seen in this area of China appears to be similar to that seen in South America but is different from those seen in Europe and North America, suggesting different evolutionary routes and selective pressures. A more comprehensive survey of the ESBL types from China is urgently needed.
Fitness Cost of Chromosomal Drug Resistance-Conferring Mutations
Authors: Sander Peter , Springer Burkhard , Prammananan Therdsak , Sturmfels Antje , Kappler Martin , Pletschette Michel , Böttger Erik C. 
Published: July 2002

ABSTRACTTo study the cost of chromosomal drug resistance mutations to bacteria, we investigated the fitness cost of mutations that confer resistance to different classes of antibiotics affecting bacterial protein synthesis (aminocyclitols, 2-deoxystreptamines, macrolides). We used a model system based on an in vitro competition assay with definedMycobacterium smegmatislaboratory mutants; selected mutations were introduced by genetic techniques to address the possibility that compensatory mutations ameliorate the resistance cost. We found that the chromosomal drug resistance mutations studied often had only a small fitness cost; compensatory mutations were not involved in low-cost or no-cost resistance mutations. When drug resistance mutations found in clinical isolates were considered, selection of those mutations that have little or no fitness cost in the in vitro competition assay seems to occur. These results argue against expectations that link decreased levels of antibiotic consumption with the decline in the level of resistance.
CTX-M-14, a Plasmid-Mediated CTX-M Type Extended-Spectrum β-Lactamase Isolated from
Escherichia coli
Authors: Ma L. , Ishii Yoshikazu , Chang Feng-Yee , Yamaguchi Keizo , Ho Monto , Siu L. K. 
Published: July 2002

ABSTRACT

Four
Escherichia coli
isolates harboring CTX-M-14, with a single Ala231→Val substitution compared to CTX-M-9, had three different ribotypes. Cefotaxime resistance was plasmid encoded and conjugatively transferable. Three isolates had the same plasmid restriction enzyme digestion profile, suggesting clonal spread of a resistant plasmid. A high
k
cat
/

K
m

value for cefotaxime (20.3 μM
−1
s
−1
) but low values for ceftazidime and aztreonam (<0.02 μM
−1
s
−1
) were observed in hydrolysis assays, indicating resistance to cefotaxime (MIC ⩾ 64 μg/ml) but susceptibility to ceftazidime (MIC ⩽ 2 μg/ml).
Genetic Diversity and Biological Control Activity of Novel Species of Closely Related Pseudomonads Isolated from Wheat Field Soils in South Australia
Authors: Ross Ian L. , Alami Younes , Harvey Paul R. , Achouak Wafa , Ryder Maarten H. 
Published: July 2002

ABSTRACT

Rhizobacteria closely related to two recently described species of pseudomonads,
Pseudomonas brassicacearum
and
Pseudomonas thivervalensis
, were isolated from two geographically distinct wheat field soils in South Australia. Isolation was undertaken by either selective plating or immunotrapping utilizing a polyclonal antibody raised against
P. brassicacearum
. A subset of 42 isolates were characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA), BIOLOG analysis, and gas chromatography-fatty acid methyl ester (GC-FAME) analysis and separated into closely related phenetic groups. More than 75% of isolates tested by ARDRA were found to have >95% similarity to either
Pseudomonas corrugata
or
P. brassicacearum-P. thivervalensis
type strains, and all isolates had >90% similarity to either type strain. BIOLOG and GC-FAME clustering showed a >70% match to ARDRA profiles. Strains representing different ARDRA groups were tested in two soil types for biological control activity against the soilborne plant pathogen
Gaeumannomyces graminis
var.
tritici
, the causative agent of take-all of wheat and barley. Three isolates out of 11 significantly reduced take-all-induced root lesions on wheat plants grown in a red-brown earth soil. Only one strain, K208, was consistent in reducing disease symptoms in both the acidic red-brown earth and a calcareous sandy loam. Results from this study indicate that
P. brassicacearum
and
P. thivervalensis
are present in Australian soils and that a level of genetic diversity exists within these two novel species but that this diversity does not appear to be related to geographic distribution. The result of the glasshouse pot trial suggests that some isolates of these species may have potential as biological control agents for plant disease.
Redirection of the Respiro-Fermentative Flux Distribution in
Saccharomyces cerevisiae
by Overexpression of the Transcription Factor Hap4p
Authors: Blom Jolanda , De Mattos M. Joost Teixeira , Grivell Leslie A. 
Published: July 2002

ABSTRACT

Reduction of aerobic fermentation on sugars by altering the fermentative/oxidative balance is of significant interest for optimization of industrial production of
Saccharomyces cerevisiae
. Glucose control of oxidative metabolism in baker's yeast is partly mediated through transcriptional regulation of the Hap4p subunit of the Hap2/3/4/5p transcriptional activator complex. To alleviate glucose repression of oxidative metabolism, we constructed a yeast strain with constitutively elevated levels of Hap4p. Genetic analysis of expression levels of glucose-repressed genes and analysis of respiratory capacity showed that Hap4p overexpression (partly) relieves glucose repression of respiration. Analysis of the physiological properties of the Hap4p overproducer in batch cultures in fermentors (aerobic, glucose excess) has shown that the metabolism of this strain is more oxidative than in the wild-type strain, resulting in a significant reduced ethanol production and improvement of growth rate and a 40% gain in biomass yield. Our results show that modification of one or more transcriptional regulators can be a powerful and a widely applicable tool for redirection of metabolic fluxes in microorganisms.
Degradation of Xylan to
d
-Xylose by Recombinant
Saccharomyces cerevisiae
Coexpressing the
Aspergillus niger
β-Xylosidase (
xlnD
) and the
Trichoderma reesei
Xylanase II (
xyn2
) Genes
Authors: La Grange D. C. , Pretorius I. S. , Claeyssens M. , van Zyl W. H. 
Published: July 2002

ABSTRACT

The β-xylosidase-encoding
xlnD
gene of
Aspergillus niger
90196 was amplified by the PCR technique from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 2,412-bp open reading frame that encodes a 804-amino-acid propeptide. The 778-amino-acid mature protein, with a putative molecular mass of 85.1 kDa, was fused in frame with the
Saccharomyces cerevisiae
mating factor α1 signal peptide (MFα1
s
) to ensure correct posttranslational processing in yeast. The fusion protein was designated Xlo2. The recombinant β-xylosidase showed optimum activity at 60°C and pH 3.2 and optimum stability at 50°C. The
K

i
(app)

value for
d
-xylose and xylobiose for the recombinant β-xylosidase was determined to be 8.33 and 6.41 mM, respectively. The
XLO2
fusion gene and the
XYN2
β-xylanase gene from
Trichoderma reesei
, located on
URA3
-based multicopy shuttle vectors, were successfully expressed and coexpressed in the yeast
Saccharomyces cerevisiae
under the control of the alcohol dehydrogenase II gene (
ADH2
) promoter and terminator. These recombinant
S. cerevisiae
strains produced 1,577 nkat/ml of β-xylanase activity when expressing only the β-xylanase and 860 nkat/ml when coexpressing the β-xylanase with the β-xylosidase. The maximum β-xylosidase activity was 5.3 nkat/ml when expressed on its own and 3.5 nkat/ml when coexpressed with the β-xylanase. Coproduction of the β-xylanase and β-xylosidase enabled
S. cerevisiae
to degrade birchwood xylan to
d
-xylose.
Interaction of Ammonium, Glucose, and Chitin Regulates the Expression of Cell Wall-Degrading Enzymes in
Trichoderma atroviride
Strain P1
Authors: Donzelli Bruno Giuliano Garisto , Harman Gary E. 
Published: July 2002

ABSTRACT

Chitinolytic and glucanolytic fungal cell wall-degrading enzymes have been suggested to be primary determinants of biocontrol by
Trichoderma
spp. We examined the effects of ammonium, glucose, chitin, and chito-oligomers on transcription of specific genes and secretion of fungal cell wall-degrading enzymes. The genes
ech42
,
nag1
, and
gluc78
were examined, as were the enzymes they encode (endochitinase CHIT42,
N
-acetylhexosaminidase CHIT73, and glucan exo-1,3-β-glucanase GLUC78, respectively).
gluc78
could be induced by nitrogen starvation alone, while both
ech42
and
nag1
required nitrogen starvation and the presence of chitin for induction. Starvation for both ammonium and glucose resulted in very early expression and secretion of all cell wall-degrading enzymes examined. In the presence of low levels of ammonium (10 mM), both chito-oligomers and chitin triggered CHIT42 and CHIT40 (chitobiosidase) production. CHIT73 secretion occurred in the presence of
N
-acetylglucosamine and chito-oligomers, while chitin was less effective. The presence of different chito-oligomers resulted in secretion of specific
N
-acetylhexosaminidases, of which CHIT73 is one. Our results indicate that the expression and secretion of cell wall-degrading enzymes is nitrogen repressed, that effects of carbon and nitrogen nutrition are interactive, and that especially for chitinolytic enzymes, the inductive effect of chitin is altered by the level of ammonium or glucose in the medium.
Quantification of Ammonia-Oxidizing Bacteria in Arable Soil by Real-Time PCR
Authors: Hermansson Anna , Lindgren Per-Eric 
Published: July 2002

ABSTRACT

Real-time PCR was used to quantify populations of ammonia-oxidizing bacteria representing the β subdivision of the class
Proteobacteria
in samples of arable soil, both nitrogen fertilized and unfertilized, from Mellby, Sweden. Primers and probes targeting a 16S ribosomal DNA region of the ammonia-oxidizing bacteria were designed and used. In the fertilized soil there were ∼6.2 × 10
7
ammonia-oxidizing bacteria per g of soil, three times more than the number of bacteria in the unfertilized soil. The lytic efficiency of bead beating in these soils was investigated by using populations of free or loosely attached bacteria, bacteria tightly bound to particles, and bacteria in nonfractionated samples. The shapes of the curves generated in these tests showed that the concentration of template DNA released at various times remained constant after 10 to 100 s of bead beating.
Plant-Derived Oils Reduce Pathogens and Gaseous Emissions from Stored Cattle Waste
Authors: Varel Vincent H. , Miller Daniel N. 
Published: July 2002

ABSTRACT

Carvacrol and thymol in combination at 6.7 mM each completely inhibited the production of short-chain volatile fatty acids and lactate from cattle waste in anoxic flasks over 23 days. Fecal coliforms were reduced from 4.6 × 10
6
to 2.0 × 10
3
cells per ml 2 days after treatment and were nondetectable within 4 days. Total anaerobic bacteria were reduced from 8.4 × 10
10
to 1.5 × 10
7
cells per ml after 2 days and continued to be suppressed to that level after 14 days. If the concentration of carvacrol or thymol were doubled (13.3 mM), either could be used to obtain the same inhibitory fermentation effect. We conclude that carvacrol or thymol may be useful as an antimicrobial chemical to control pathogens and odor in stored livestock waste.
Isoenzyme Multiplicity and Characterization of Recombinant Manganese Peroxidases from
Ceriporiopsis subvermispora
and
Phanerochaete chrysosporium
Authors: Larrondo Luis F. , Lobos Sergio , Stewart Phillip , Cullen Dan , Vicuña Rafael 
Published: July 2002

ABSTRACT

We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes
Ceriporiopsis subvermispora
(MnP1) and
Phanerochaete chrysosporium
(H4) under control of the α-amylase promoter from
Aspergillus oryzae
in
Aspergillus nidulans
. The recombinant proteins (rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from
P. chrysosporium
(pI 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from
C. subvermispora
grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize
o
-dianisidine and
p
-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from
P. chrysosporium
.