bluuebunny/crossref_metadata_2025_split huggingface dataset:
Predicting Evolution by In Vitro Evolution Requires Determining Evolutionary Pathways
Authors: Hall Barry G. 
Published: October 2002

ABSTRACT

In an early example of DNA shuffling, Stemmer (W. P. C. Stemmer, Nature
370:
389-390, 1994) demonstrated a dramatic improvement in the activity of the TEM-1 β-lactamase toward cefotaxime as the consequence of six amino acid substitutions. It has been pointed out (B. G. Hall, FEMS Microbiol. Lett.
178:
1-6, 1999; M. C. Orencia, J. S. Yoon, J. E. Ness, W. P. Stemmer, and R. C. Stevens, Nat. Struct. Biol.
8:
238-242, 2001) that the power of DNA shuffling might be applied to the problem of predicting evolution in nature from in vitro evolution in the laboratory. As a predictor of natural evolutionary processes, that power may be misleading because in nature mutations almost always arise one at a time, and each advantageous mutation must be fixed into the population by an evolutionary pathway that leads from the wild type to the fully evolved sequence. Site-directed mutagenesis was used to introduce each of Stemmer's six substitutions into TEM-1, the best single mutant was chosen, and each of the remaining five substitutions was introduced. Repeated rounds of site-directed mutagenesis and selection of the best mutant were used in an attempt to construct a pathway between the wild-type TEM-1 and Stemmer's mutant with six mutations. In the present study it is shown (i) that no such pathway exists between the wild-type TEM-1 and the supereffective cefotaxime-hydrolyzing mutant that was generated by six amino acid substitutions via DNA shuffling (Stemmer, Nature
370:
389-390, 1994) but that a pathway to a fourfold more efficient enzyme resulting from four of the same substitutions does exist, and (ii) that the more efficient enzyme is likely to arise in nature as the result of a single mutation in the naturally occurring TEM-52 allele.
In Vitro Activities of Garenoxacin (BMS-284756) against 170 Clinical Isolates of Nine
Pasteurella
Species
Authors: Goldstein Ellie J. C. , Citron Diane M. , Merriam C. Vreni , Warren Yumi A. , Tyrrell Kerin L. , Fernandez Helen T. 
Published: October 2002

ABSTRACT

The in vitro susceptibilities of 170 clinical isolates plus 12 American Type Culture Collection strains of
Pasteurella
species comprising nine species and three
Pasteurella multocida
subspecies were studied by an agar dilution method. Garenoxacin (BMS-284756), a new des-fluoro(6) quinolone, was active at ≤0.06 μg/ml against all isolates, including four β-lactamase-producing strains, with >90% of the strains susceptible to ≤0.008 μg/ml. Garenoxacin was generally 1 to 2 dilutions more active than levofloxacin and moxifloxacin and was the most active agent tested. Cefoxitin required 1 μg/ml for inhibition of 51 of 182 (29%) of strains, and 3 strains (also β-lactamase producers) were resistant to doxycycline.
Prevalence of Protease and Reverse Transcriptase Drug Resistance Mutations over Time in Drug-Naïve Human Immunodeficiency Virus Type 1-Positive Individuals in Rio de Janeiro, Brazil
Authors: Dumans Ana T. , Soares Marcelo A. , Pieniazek Danuta , Kalish Marcia L. , De Vroey Veronique , Hertogs Kurt , Tanuri Amilcar 
Published: October 2002

ABSTRACT
The prevalence of mutations that confer resistance to protease inhibitors and to nucleoside and nonnucleoside reverse transcriptase inhibitors in 49 blood samples from drug-naïve human immunodeficiency virus type 1-infected blood donors living in Rio de Janeiro state, Brazil, in 1998 was evaluated genotypically and phenotypically.
Voriconazole Inhibition of the Metabolism of Tacrolimus in a Liver Transplant Recipient and in Human Liver Microsomes
Authors: Venkataramanan Raman , Zang Shimin , Gayowski Timothy , Singh Nina 
Published: October 2002

ABSTRACT
The purpose of this study was to assess the effect of voriconazole on the blood tacrolimus concentration in a liver transplant recipient and to examine the interaction between voriconazole and tacrolimus by using human liver microsomes. Two subjects were enrolled in the clinical study: one received voriconazole, and the other received a placebo. Tacrolimus metabolism was evaluated in human liver microsomes at various concentrations in the absence and presence of various concentrations of voriconazole. Coadministration of voriconazole and tacrolimus resulted in elevated (nearly 10-fold-higher) trough tacrolimus blood concentrations in the liver transplant patient. In the in vitro study, voriconazole at a concentration of 10.4 ± 4.3 μg/ml inhibited the metabolism of tacrolimus by 50%. Clinically relevant concentrations of voriconazole inhibited the metabolism of tacrolimus in human liver microsomes. Close monitoring of the blood concentration and adjustment in the dose of tacrolimus are warranted in transplant recipients treated with voriconazole.
Effect of the Echinocandin Caspofungin on Expression of
Candida albicans
Secretory Aspartyl Proteinases and Phospholipase In Vitro
Authors: Ripeau Jean-Sébastien , Aumont Francine , Belhumeur Pierre , Ostrosky-Zeichner Luis , Rex John H. , de Repentigny Louis 
Published: October 2002

ABSTRACT

Although the echinocandin caspofungin primarily inhibits the synthesis of cell wall 1,3-β-
d
-glucan, its fungicidal activity could also potentially perturb the expression of virulence factors involved in the ability of
Candida albicans
to cause infection. Expression of the
C. albicans
secretory aspartyl proteinase (
SAP
) and phospholipase B (
PLB
) virulence genes was determined by reverse transcription-PCR after the addition of caspofungin to cells grown for 15 h in Sabouraud dextrose broth. In cells that remained viable, expression of
SAP1
to
SAP3
,
SAP7
to
SAP9,
and
PLB1
was unaltered after exposure to fungicidal concentrations (4 to 16 μg/ml) of caspofungin over a period of 7 h. However, expression of
SAP5
increased steadily beginning 1 h after exposure to caspofungin. These results indicate that caspofungin is rapidly fungicidal against
C. albicans
, before any suppression of
SAP
or
PLB1
gene expression can occur.
Nonfunctionality of
Aspergillus sojae aflR
in a Strain of
Aspergillus parasiticus
with a Disrupted
aflR
Gene
Authors: Takahashi Tadashi , Chang Perng-Kuang , Matsushima Kenichiro , Yu Jiujiang , Abe Keietsu , Bhatnagar Deepak , Cleveland Thomas E. , Koyama Yasuji 
Published: October 2002

ABSTRACT

Aspergillus sojae
belongs to the
Aspergillus
section
Flavi
but does not produce aflatoxins. The functionality of the
A. sojae aflR
gene (
aflRs
) was examined by transforming it into an Δ
aflR
strain of
A. parasiticus
, derived from a nitrate-nonutilizing, versicolorin A (VERA)-accumulating strain. The
A. parasiticus aflR
gene (
aflRp
) transformants produced VERA, but the
aflRs
transformants did not. Even when
aflRs
was placed under the control of the amylase gene (
amyB
) promoter of
Aspergillus oryzae
, the
amy
(
p
)::
aflRs
transformants did not produce VERA. A chimeric construct containing the
aflRs
promoter plus the
aflRs
N- and
aflRp
C-terminal coding regions could restore VERA production, but a construct containing the
aflRp
promoter plus the
aflRp
N- and
aflRs
C-terminal coding regions could not. These results show that the
A. sojae aflR
promoter is functional in
A. parasiticus
and that the HAHA motif does not affect the function of the resulting hybrid AflR. We conclude that the lack of aflatoxin production by
A. sojae
can be attributed, at least partially, to the premature termination defect in
aflRs
, which deletes the C-terminal transcription activation domain that is critical for the expression of aflatoxin biosynthetic genes.
Molecular Epidemiology of
Clostridium perfringens
Related to Food-Borne Outbreaks of Disease in Finland from 1984 to 1999
Authors: Lukinmaa Susanna , Takkunen Elina , Siitonen Anja 
Published: October 2002

ABSTRACT

From 1975 to 1999,
Clostridium perfringens
caused 238 food-borne disease outbreaks in Finland, which is 20% of all such reported outbreaks during these years. The fact that
C. perfringens
is commonly found in human and animal stools and that it is also widespread in the environment is a disadvantage when one is searching for the specific cause of a food-borne infection by traditional methods. In order to strengthen the evidence-based diagnostics of food poisonings suspected to be caused by
C. perfringens
, we retrospectively investigated 47
C. perfringens
isolates by PCR for the
cpe
gene, which encodes enterotoxin; by reversed passive latex agglutination to detect the enterotoxin production; and by pulsed-field gel electrophoresis (PFGE) to compare their genotypes after restriction of DNA by the enzymes
Sma
I and
Apa
I. The strains were isolated during 1984 to 1999 from nine food-borne outbreaks of disease originally reported as having been caused by
C. perfringens
. In seven of the nine outbreaks our results supported the fact that the cause was
C. perfringens
. Our findings emphasize the importance of a more detailed characterization of
C. perfringens
isolates than mere identification to the species level in order to verify the cause of an outbreak. Also, to increase the probability of finding the significant
cpe
-positive
C. perfringens
strains, it is very important to isolate and investigate more than one colony from the fecal culture of a patient and screen all these isolates for the presence of the
cpe
gene before further laboratory work is done.
Real-Time PCR Quantification of
rbcL
(Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase) mRNA in Diatoms and Pelagophytes
Authors: Wawrik B. , Paul J. H. , Tabita F. R. 
Published: October 2002

ABSTRACT

Transcriptional activity is often used as a surrogate for gene expression in environmental microbial communities. We developed a real-time PCR assay in which the ABI-Prism (PE Applied Biosystems) detection system is used for quantification of large-subunit ribulose-1,5-bisphosphate caboxylase/oxygenase (
rbcL
) mRNA in diatoms and pelagophytes both in cultures and from natural phytoplankton communities. Plasmid DNA containing
rbcL
inserts, as well as in vitro transcribed mRNA of the plasmids, was used to generate standard curves with a dynamic range of more than 6 orders of magnitude with high accuracy and precision (
R
2
= 0.998). Expression levels in a cultured diatom (
Phaeodactylum tricornutum
) were quantified through one light-dark cycle by using traditional
35
S-labeled oligonucleotide hybridization and real-time PCR. The mRNA levels detected by the two techniques were similar and correlated well (
R
2
= 0.95; slope = 1.2). The quantities obtained by hybridization were slightly, yet significantly, larger (
t
= 5.29;
P
= 0.0011) than the quantities obtained by real-time PCR. This was most likely because partially degraded transcripts were not detected by real-time PCR.
rbcL
mRNA detection by real-time PCR was 3 orders of magnitude more sensitive than
rbcL
mRNA detection by hybridization. Diatom and pelagophyte
rbcL
mRNAs were also quantified in a profile from an oligotrophic site in the Gulf of Mexico. We detected the smallest amount of diatom
rbcL
expression in the surface water and maximum expression at a depth that coincided with the depth of the subsurface chlorophyll maximum. These results indicate that real-time PCR may be utilized for quantification of microbial gene expression in the environment.
Factors Controlling Anaerobic Ammonium Oxidation with Nitrite in Marine Sediments
Authors: Dalsgaard Tage , Thamdrup Bo 
Published: October 2002

ABSTRACT

Factors controlling the anaerobic oxidation of ammonium with nitrate and nitrite were explored in a marine sediment from the Skagerrak in the Baltic-North Sea transition. In anoxic incubations with the addition of nitrite, approximately 65% of the nitrogen gas formation was due to anaerobic ammonium oxidation with nitrite, with the remainder being produced by denitrification. Anaerobic ammonium oxidation with nitrite exhibited a biological temperature response, with a rate optimum at 15°C and a maximum temperature of 37°C. The biological nature of the process and a 1:1 stoichiometry for the reaction between nitrite and ammonium indicated that the transformations might be attributed to the anammox process. Attempts to find other anaerobic ammonium-oxidizing processes in this sediment failed. The apparent

K
m

of nitrite consumption was less than 3 μM, and the relative importance of ammonium oxidation with nitrite and denitrification for the production of nitrogen gas was independent of nitrite concentration. Thus, the quantitative importance of ammonium oxidation with nitrite in the jar incubations at elevated nitrite concentrations probably represents the in situ situation. With the addition of nitrate, the production of nitrite from nitrate was four times faster than its consumption and therefore did not limit the rate of ammonium oxidation. Accordingly, the rate of this process was the same whether nitrate or nitrite was added as electron acceptor. The addition of organic matter did not stimulate denitrification, possibly because it was outcompeted by manganese reduction or because transport limitation was removed due to homogenization of the sediment.
Biochemical and Genetic Characterization of Mundticin KS, an Antilisterial Peptide Produced by
Enterococcus mundtii
NFRI 7393
Authors: Kawamoto Shinichi , Shima Jun , Sato Rumi , Eguchi Tomoko , Ohmomo Sadahiro , Shibato Junko , Horikoshi Naoko , Takeshita Kazuko , Sameshima Takashi 
Published: October 2002

ABSTRACT

Mundticin KS, a bacteriocin produced by
Enterococcus mundtii
NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen
Listeria monocytogenes
. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (
mun
locus) for mundticin KS production was cloned, and DNA sequencing revealed that the
mun
locus consists of three genes, designated
munA
,
munB
, and
munC
. The
munA
gene encodes a 58-amino-acid mundticin KS precursor,
munB
encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and
munC
encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by
E. mundtii
ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in
Escherichia coli
as recombinant peptides and found to be different in activity against certain
Lactobacillus
strains, such as
Lactobacillus plantarum
and
Lactobacillus curvatus
. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the
mun
locus in heterogeneous hosts such as
E. faecium
,
L. curvatus
, and
Lactococcus lactis
. Based on our results, the
mun
locus is located on a 50-kb plasmid, pML1, of
E. mundtii
NFRI 7393.
Metabolic Engineering of a Novel Propionate-Independent Pathway for the Production of Poly(3-Hydroxybutyrate-
co
-3-Hydroxyvalerate) in Recombinant
Salmonella enterica
Serovar Typhimurium
Authors: Aldor Ilana S. , Kim Seon-Won , Prather Kristala L. Jones , Keasling Jay D. 
Published: October 2002

ABSTRACT

A pathway was metabolically engineered to produce poly(3-hydroxybutyrate-
co
-3-hydroxyvalerate) (PHBV), a biodegradable thermoplastic with proven commercial applications, from a single, unrelated carbon source. An expression system was developed in which a
prpC
strain of
Salmonella enterica
serovar Typhimurium, with a mutation in the ability to metabolize propionyl coenzyme A (propionyl-CoA), served as the host for a plasmid harboring the
Acinetobacter
polyhydroxyalkanoate synthesis operon (
phaBCA
) and a second plasmid with the
Escherichia coli sbm
and
ygfG
genes under an independent promoter. The
sbm
and
ygfG
genes encode a novel (2
R
)-methylmalonyl-CoA mutase and a (2
R
)-methylmalonyl-CoA decarboxylase, respectively, which convert succinyl-CoA, derived from the tricarboxylic acid cycle, to propionyl-CoA, an essential precursor of 3-hydroxyvalerate (HV). The
S. enterica
system accumulated PHBV with significant HV incorporation when the organism was grown aerobically with glycerol as the sole carbon source. It was possible to vary the average HV fraction in the copolymer by adjusting the arabinose or cyanocobalamin (precursor of coenzyme B
12
) concentration in the medium.
Adhesion to Bile Drain Materials and Physicochemical Surface Properties of
Enterococcus faecalis
Strains Grown in the Presence of Bile
Authors: Waar Karola , van der Mei Henny C. , Harmsen Hermie J. M. , Degener John E. , Busscher Henk J. 
Published: October 2002

ABSTRACT

The aim of this study is to determine whether growth in the presence of bile influences the surface properties and adhesion to hydrophobic bile drain materials of
Enterococcus faecalis
strains expressing aggregation substance (Agg) or enterococcal surface protein (Esp), two surface proteins that are associated with infections. After growth in the presence of bile, the strains were generally more hydrophobic by water contact angles and the zeta potentials were more negative than when the strains were grown in the absence of bile. Nitrogen was found in lower surface concentrations upon growth in the presence of bile, whereas higher surface concentrations of oxygen were measured by X-ray photoelectron spectroscopy. Moreover, an up to twofold-higher number of bacteria adhered after growth in bile for
E. faecalis
not expressing Agg or Esp and
E. faecalis
with Esp on its surface.
E. faecalis
expressing Agg did not adhere in higher numbers after growth in bile, possibly because they mainly adhere through positive cooperativity and less through direct interactions with a substratum surface. Since adhesion of bacteria is the first step in biomaterial-centered infection, it can be concluded that growth in bile increases the virulence of
E. faecalis
.
Detection of both Hepatitis A Virus and Norwalk-Like Virus in Imported Clams Associated with Food-Borne Illness
Authors: Kingsley David H. , Meade Gloria K. , Richards Gary P. 
Published: October 2002

ABSTRACT
Hepatitis A virus (HAV) and Norwalk-like virus (NLV) were detected by reverse transcription-PCR in clams imported into the United States from China. An epidemiological investigation showed that these clams were associated with five cases of Norwalk-like gastroenteritis in New York State in August 2000 (Food and Drug Administration Import Alert 16-50). They were labeled “cooked” but appeared raw. Viral RNA extraction was performed by using dissected digestive tissues rather than whole shellfish meats; this was followed by glycine buffer elution, polyethylene glycol precipitation, Tri-Reagent treatment, and purification of poly(A) RNA with magnetic beads coupled to poly(dT) oligonucleotides. We identified HAV and NLV as genotype I and genogroup II strains, respectively. Both viruses have high levels of homology to Asian strains. An analysis of fecal coliforms revealed a most-probable number of 93,000/100 g of clam meat, which is approximately 300-fold higher than the hygienic standard for shellfish meats.
Extremely Stable and Versatile Carboxylesterase from a Hyperthermophilic Archaeon
Authors: Hotta Yuji , Ezaki Satoshi , Atomi Haruyuki , Imanaka Tadayuki 
Published: October 2002

ABSTRACT

We have found that the hyperthermophilic archaeon
Pyrobaculum calidifontis
VA1 produced a thermostable esterase. We isolated and sequenced the esterase gene (

est
Pc

) from strain VA1.

est
Pc

consisted of 939 bp, corresponding to 313 amino acid residues with a molecular mass of 34,354 Da. As

est
Pc

showed significant identity (30%) to mammalian hormone-sensitive lipases (HSLs), esterase of
P. calidifontis
(Est) could be regarded as a new member of the HSL family. Activity levels of the enzyme were comparable or higher than those of previously reported enzymes not only at high temperature (6,410 U/mg at 90°C), but also at ambient temperature (1,050 U/mg at 30°C). The enzyme displayed extremely high thermostability and was also stable after incubation with various water-miscible organic solvents at a concentration of 80%. The enzyme also exhibited activity in the presence of organic solvents. Est of
P. calidifontis
showed higher hydrolytic activity towards esters with short to medium chains, with
p
-nitrophenyl caproate (C
6
) the best substrate among the
p
-nitrophenyl esters examined. As for the alcoholic moiety, the enzyme displayed esterase activity towards esters with both straight- and branched-chain alcohols. Most surprisingly, we found that this Est enzyme hydrolyzed the tertiary alcohol ester
tert
-butyl acetate, a feature very rare among previously reported lipolytic enzymes. The extreme stability against heat and organic solvents, along with its activity towards a tertiary alcohol ester, indicates a high potential for the Est of
P. calidifontis
in future applications.
Cyclic AMP and Acyl Homoserine Lactones Increase the Cultivation Efficiency of Heterotrophic Bacteria from the Central Baltic Sea
Authors: Bruns Alke , Cypionka Heribert , Overmann Jörg 
Published: October 2002

ABSTRACT

The effect of signal molecules on the cultivation efficiency of bacteria from the Gotland Deep in the central Baltic Sea was investigated. Numbers of cultivated cells were determined by the most-probable-number (MPN) technique. Artificial brackish water supplemented with different carbon substrates at low concentrations (200 μM each) was employed as the growth medium. Compared to the results of previous studies, this approach yielded significantly higher cultivation efficiencies (up to 11% in fluid media). A further and pronounced increase in cultivation success was accomplished by the addition of cyclic AMP (cAMP),
N
-butyryl homoserine lactone, or
N
-oxohexanoyl-
dl
-homoserine lactone at a low concentration of 10 μM. The most effective inducer was cAMP, which led to cultivation efficiencies of up to 100% of total bacterial counts. From the highest positive dilutions of these latter MPN series, several strains were isolated in pure culture and one strain (G100) was used to study the physiological effect of cAMP. Dot blot hybridization revealed, however, that strain G100 represented only a small fraction of the total bacterial community. This points towards an inherent limitation of the MPN approach, which does not necessarily recover abundant species from highly diverse communities. Bacterial cells of strain G100 that were starved for 6 weeks attained a higher growth rate and a higher biomass yield when resuscitated in the presence of cAMP instead of AMP.
Sodium Dodecyl Sulfate Hypersensitivity of
clpP
and
clpB
Mutants of
Escherichia coli
Authors: Rajagopal Soumitra , Sudarsan Narasimhan , Nickerson Kenneth W. 
Published: October 2002

ABSTRACT

We studied the hypersensitivity of
clpP
and
clpB
mutants of
Escherichia coli
to sodium dodecyl sulfate (SDS). Both wild-type
E. coli
MC4100 and
lon
mutants grew in the presence of 10% SDS, whereas isogenic
clpP
and
clpB
single mutants could not grow above 0.5% SDS and
clpA
and
clpX
single mutants could not grow above 5.0% SDS. For wild-type
E. coli
, cellular ClpP levels as determined by Western immunoblot analysis increased ca. sixfold as the levels of added SDS increased from 0 to 2%. Capsular colanic acid, measured as uronic acid, increased ca. sixfold as the levels of added SDS increased from 2 to 10%. Based on these findings, 3 of the 19 previously identified SDS shock proteins (M. Adamowicz, P. M. Kelley, and K. W. Nickerson, J. Bacteriol. 173:229-233, 1991) are tentatively identified as ClpP, ClpX, and ClpB.