bluuebunny/crossref_metadata_2025 huggingface dataset:
Maintenance of Genome Stability in
Saccharomyces cerevisiae
Authors: Kolodner Richard D. , Putnam Christopher D. , Myung Kyungjae
Published: October 2002

Most human cancer cells show signs of genome instability, ranging from elevated mutation rates to gross chromosomal rearrangements and alterations in chromosome number. Little is known about the molecular mechanisms that generate this instability or how it is suppressed in normal cells. Recent studies of the yeast
Saccharomyces cerevisiae
have begun to uncover the extensive and redundant pathways that keep the rate of genome rearrangements at very low levels. These studies, which we review here, have implicated more than 50 genes in the suppression of genome instability, including genes that function in S-phase checkpoints, recombination pathways, and telomere maintenance. Human homologs of several of these genes have well-established roles as tumor suppressors, consistent with the hypothesis that the mechanisms preserving genome stability in yeast are the same mechanisms that go awry in cancer.
Surface-Responsive Materials
Authors: Russell T. P.
Published: October 2002

Synthetic polymers offer a wealth of opportunities to design responsive materials triggered by external stimuli. Changing the length, chemical composition, architecture, and topology of the chains allows response mechanisms and rates to be easily manipulated; and devices based on the entropy of the chains, surface energies, and specific segmental interactions can readily be made. Although numerous applications exist, intriguing possibilities are emerging that have tremendous potential to further developments in surface-responsive materials.
Connecting Chromosomes, Crisis, and Cancer
Authors: Maser Richard S. , DePinho Ronald A.
Published: October 2002

Cancer is a disease of impaired genome stability. The molecular forces that maintain genome integrity and sense altered chromosome structure are invariably subverted in cancer cells. Here, we explore the contrasting contributions of telomeres in the initiation and suppression of cancer and review the evidence supporting a role for telomere dysfunction as a mechanism driving the radical chromosomal aberrations that typify cancer genomes. Recent work suggests that passage of cells through crisis in the setting of deactivated DNA damage checkpoints provides a mutational mechanism that can generate the diverse genetic alterations required for cancer initiation. A greater understanding of telomere-induced crisis and the cell's crisis management mechanisms should guide the rational development of new therapeutics for cancer and other disorders.
Heterologous Expression of Epothilone Biosynthetic Genes in
Myxococcus xanthus
Authors: Julien Bryan , Shah Sanjay
Published: October 2002

Epothilones are potential anticancer drugs that stabilize microtubules in a manner similar to paclitaxel (Taxol). Epothilones are produced from the myxobacterium
Sorangium cellulosum
, which has a 16-h doubling time and produces only milligram-per-liter amounts of epothilone A and epothilone B. Furthermore, genetic manipulation of
S. cellulosum
is difficult. To produce epothilones in a more genetically amenable and rapidly growing host, we chose the closely related and best-characterized myxobacteria
Myxococcus xanthus
. We inserted 65.4 kb of
S. cellulosum
DNA that encompassed the entire epothilone gene cluster into the chromosome of
M. xanthus
by a series of homologous recombination events. The resulting strain produced epothilones A and B. Construction of a strain that contained a mutation in
epoK
, the P450 epoxidase, resulted in production of epothilones C and D.
TheCandida dubliniensis CdCDR1Gene Is Not Essential for Fluconazole Resistance
Authors: Moran Gary , Sullivan Derek , Morschhäuser Joachim , Coleman David
Published: October 2002

The present study investigated the role of theCandida dubliniensis CdCDR1andCdCDR2genes in the development of fluconazole resistance. TheC. dubliniensis CdCDR1gene was 92% identical at the nucleotide sequence level to the correspondingC. albicansgene. However, 58% (14 of 24) ofC. dubliniensisgenotype 1 isolates tested harbored a nonsense mutation in theCdCDR1open reading frame that converted codon 756 (TAT) to a TAG translational stop codon. Analysis of five of theseC. dubliniensisisolates by Western immunoblotting showed that they expressed a truncated 85-kDa CdCdr1p compared to the full-length 170-kDa CdCdr1p. Expression ofCdCDR1alleles from sixC. dubliniensisisolates in apdr5 Saccharomyces cerevisiaestrain revealed thatCdCDR1alleles from three isolates that encoded truncated proteins were unable to confer resistance to drugs and antifungals. However, reassignment of the TAG sequence at codon 756 to TAT (encoding tyrosine) in an allele from strain CD36 conferred the ability to mediate resistance to multiple drugs. Fluconazole-resistant isolates ofC. dubliniensisharboring functional alleles ofCdCDR1were found to exhibit two- to ninefold-higher levels ofCdCDR1mRNA than did matched fluconazole-susceptible isolates. By comparison, levels ofCdMDR1expression ranged from approximately 50- to 100-fold greater in resistant isolates. Fluconazole resistance was also identified in isolates harboring nonfunctionalCdCDR1alleles, but resistance in these isolates was only associated with increasedCdMDR1expression. Targeted disruption of two functional alleles ofCdCDR1in a fluconazole-resistant derivative ofC. dubliniensisthat overexpressed bothCdCDR1andCdMDR1revealed that althoughCdCDR1was important for mediating reduced susceptibility to itraconazole and ketoconazole, there was no affect on fluconazole susceptibility in the double mutant. Evidence presented in this study reveals thatCdCDR1is not essential for the development of fluconazole resistance inC. dubliniensis.
Effect of 5-Iodo-2′-Deoxyuridine on Vaccinia Virus (Orthopoxvirus) Infections in Mice
Authors: Neyts Johan , Verbeken Erik , De Clercq Erik
Published: October 2002

There is a concern that there may be unregistered stocks of smallpox that can be used for bioterrorism or biological warfare. According to the WHO Advisory Committee on Variola Research, there is a need to develop strategies to treat smallpox infections should they reappear. It would also be important to have an effective drug at hand for the treatment of monkeypox disease in humans. We show here that 5-iodo-2′-deoxyuridine (IDU) is a potent inhibitor of vaccinia virus (VV) replication and that IDU inhibits VV DNA synthesis in a dose-dependent way. The in vivo protective effect of IDU was assessed in the VV tail lesion model in immunocompetent mice and in a lethal model for VV infection in SCID (severe combined immune deficiency) mice that had been infected either intranasally, intraperitoneally, or intravenously. Subcutaneous treatment with IDU at 150 and 100 mg/kg of body weight markedly reduced the number of tail lesions in immunocompetent NMRI mice. Untreated intranasally VV-infected SCID mice died at 20.8 ± 3.1 days after infection (mean ± standard deviation). Treatment with IDU (subcutaneously, 150 mg/kg/day [from day 0 to 4] and 75 mg/kg/day [from day 6 to 11]) delayed-virus induced mortality by 15 days (mean day of death ± standard deviation, 35.8 ± 6.7;
P
< 0.0001). This protective effect was associated with (i) an improvement of lung histology and (ii) a marked reduction in lung viral titers. IDU also delayed VV-induced mortality when mice had either been infected intraperitoneally or intravenously. Even when the start of treatment with IDU (in intraperitoneally VV-infected mice) was postponed until 2 or 4 days after infection, an important delay in virus-induced mortality was noted.
Ertapenem versus Ceftriaxone Followed by Appropriate Oral Therapy for Treatment of Complicated Urinary Tract Infections in Adults: Results of a Prospective, Randomized, Double-Blind Multicenter Study
Authors: Tomera Kevin M. , Burdmann Emmanuel A. , Reyna Oscar G. Pamo , Jiang Qi , Wimmer Wendy M. , Woods Gail L. , Gesser Richard M.
Published: October 2002

The efficacy and safety of intravenous (i.v.) ertapenem (1 g once a day) with the option to switch to an oral agent for treatment of adults with complicated urinary tract infections (UTIs) were compared with that of i.v. ceftriaxone (1 g daily) with the same oral switch option in a multicenter, double-blind, prospective, randomized study. At entry, 592 patients were assigned to one of two strata: acute pyelonephritis or other complicated UTI without acute pyelonephritis. After a minimum of 3 days, patients could be switched to an oral antimicrobial agent. A total of 159 patients in the ertapenem group and 171 patients in the ceftriaxone group were microbiologically evaluable. Approximately 95% of patients in each treatment group were switched to oral therapy. The most common pathogens were
Escherichia coli
and
Klebsiella pneumoniae
. At the primary efficacy endpoint 5 to 9 days after treatment, 91.8% of patients who received ertapenem and 93.0% of those who received ceftriaxone had a favorable microbiological response (95% confidence interval for the difference, adjusting for strata, −7.6 to 5.1%), indicating that outcomes in the two treatment groups were equivalent. Microbiological success rates for the two treatment groups were similar when compared by stratum and also by severity of infection. The frequency and severity of drug-related adverse events were generally similar in both treatment groups. In this study, ertapenem was as effective as ceftriaxone for the initial treatment of complicated UTIs in adults, was generally well tolerated, and had a similar overall safety profile.
Characterization of Sparsomycin Resistance in
Streptomyces sparsogenes
Authors: Lázaro E. , Sanz E. , Remacha M. , Ballesta J. P. G.
Published: October 2002

The antitumor antibiotic sparsomycin, produced by
Streptomyces sparsogenes,
is a universal translation inhibitor that blocks the peptide bond formation in ribosomes from all species. Sparsomycin-resistant strains were selected by transforming the sensitive
Streptomyces lividans
with an
S. sparsogenes
library. Resistance was linked to the presence of a plasmid containing an
S. sparsogenes
5.9-kbp DNA insert. A restriction analysis of the insert traced down the resistance to a 3.6-kbp DNA fragment, which was sequenced. The analysis of the fragment nucleotide sequence together with the previous restriction data associate the resistance to
srd
, an open reading frame of 1,800 nucleotides. Ribosomes from
S. sparsogenes
and the
S. lividans
-resistant strains are equally sensitive to the inhibitor and bind the drug with similar affinity. Moreover, the drug was not modified by the resistant strains. However, resistant cells accumulated less antibiotic than the sensitive ones. In addition, membrane fractions from the resistant strains showed a higher capacity for binding the drug. The results indicate that resistance in the producer strain is not connected to either ribosome modification or drug inactivation, but it might be related to an alteration in the sparsomycin permeability barrier.
Consumption of Imipenem Correlates with β-Lactam Resistance in
Pseudomonas aeruginosa
Authors: Lepper Philipp M. , Grusa Eberhard , Reichl Helga , Högel Josef , Trautmann Matthias
Published: October 2002

It is generally assumed that the antibiotic prescription policy of a hospital has a significant impact on bacterial resistance rates; however, few studies are available to support this concept with valid statistical data. During a 3-year period from 1997 to 2000, we monitored the consumption of β-lactam and other antibiotics with known activity against
Pseudomonas aeruginosa
in a 600-bed community hospital. Monthly isolations of
P. aeruginosa
were assessed, and resistance rates were recorded. Partial correlation coefficients between consumption and resistance rates were determined, taking into account possible associations with other variables such as seasonal effects and transfers from other hospitals. A total of 30 ± 7 novel
P. aeruginosa
strains per month were isolated without epidemic clustering. Prescriptions of imipenem varied significantly during the study period, while prescriptions of other antipseudomonal agents were stable, with the exception of an increase in piperacillin-tazobactam prescriptions. Rates of resistance of
P. aeruginosa
to the antimicrobial agents used showed a time course similar to figures for imipenem consumption. Monthly rates of resistance to imipenem (partial correlation coefficient [cc], 0.63), piperacillin-tazobactam (cc, 0.57), and ceftazidime (cc, 0.56) were significantly associated with imipenem prescription rates in the same or the preceding month, while consumption of ceftazidime or piperacillin-tazobactam had no apparent association with resistance. Among the variables investigated, imipenem consumption was identified as the major factor associated with both carbapenem and β-lactam resistance in endemic
P. aeruginosa
. Periods of extensive imipenem use were associated with significant increases in resistance. Our data support the concept that a written antibiotic policy which balances the use of various antibiotic classes may help to avoid disturbances of a hospital's microbial sensitivity patterns.
Human Immunodeficiency Virus Type 1 Genotypic and Pharmacokinetic Determinants of the Virological Response to Lopinavir-Ritonavir-Containing Therapy in Protease Inhibitor-Experienced Patients
Authors: Masquelier Bernard , Breilh Dominique , Neau Didier , Lawson-Ayayi Sylvie , Lavignolle Valérie , Ragnaud Jean-Marie , Dupon Michel , Morlat Philippe , Dabis F. , Fleury H.
Published: October 2002

The response to regimens including lopinavir-ritonavir (LPV/r) in patients who have received multiple protease (PR) inhibitors (PI) can be analyzed in terms of human immunodeficiency virus type 1 (HIV-1) genotypic and pharmacokinetic (pK) determinants. We studied these factors and the evolution of HIV-1 resistance in response to LPV/r in a prospective study of patients receiving LPV/r under a temporary authorization in Bordeaux, France. HIV-1 PR and reverse transcriptase sequences were determined at baseline LPV/r for all the patients and at month 3 (M3) and M6 in the absence of response to treatment. pK measurements were determined at M1 and M3. Virological failure (VF) was defined as a plasma viral load ≥400 copies/ml at M3. A multivariate analysis of the predictors of VF, including clinical and biological characteristics and the treatment history of the patients, was performed. The PR gene sequence at M0, including individual mutations or a previously defined LPV mutation score (D. J. Kempf, J. D. Isaacson, M. S. King, S. C. Brun, Y. Xu, K. Real, B. M. Bernstein, A. J. Japour, E. Sun, and R. A. Rode, J. Virol.
75:
7262-7269, 2001), and the individual exposure to LPV were also included covariates. Sixty-eight patients were enrolled. Thirty-four percent had a virological response at M3. An LPV mutation score of >5 mutations, the presence of the PR I54V mutation at baseline, a high number of previous PIs, prior therapy with ritonavir or indinavir, absence of coprescription of efavirenz, and a lower exposure to LPV or lower LPV trough concentrations were independently associated with VF on LPV/r. Additional PI resistance mutations, including primary mutation I50V, could be selected in patients failing on LPV/r. Genotypic and pK parameters should be used to optimize the virological response to LPV/r in PI-experienced patients and to avoid further viral evolution.
Application of Real-Time PCR for Determination of Antiviral Drug Susceptibility of Herpes Simplex Virus
Authors: Stránská Růžena , van Loon Anton M. , Polman Merjo , Schuurman Rob
Published: October 2002

A quantitative real-time PCR (TaqMan) assay was developed for determination of antiviral drug susceptibility of herpes simplex virus (HSV). After short-time culture of the virus, the antiviral drug susceptibility of HSV isolates for acyclovir (ACV) was determined by measuring the reduction of the HSV type 1 (HSV-1) DNA levels in culture supernatants using real-time PCR. The 50% inhibitory concentration was reported as the concentration of antiviral drug that reduced the number of HSV-1 DNA copies by 50%. A total of 15 well-characterized ACV-sensitive or -resistant strains and clinical isolates were used for assay evaluation. The new assay with real-time PCR readout permitted rapid (3 days), objective, and reproducible determination of HSV-1 drug susceptibilities with no need for stringent control of initial multiplicity of infection. Furthermore, the real-time PCR assay results showed good correlation (
r
= 0.86) with those for the plaque reduction assay. In conclusion, the real-time PCR assay described here is a suitable quantitative method for determination of antiviral susceptibility of HSV-1, amenable for use in the routine diagnostic virology laboratory.
Occurrence of Antibiotic Resistance Gene Cassettes
aac
(
6
′)
-Ib
,
dfrA5
,
dfrA12
, and
ereA2
in Class I Integrons in Non-O1, Non-O139
Vibrio cholerae
Strains in India
Authors: Thungapathra M. , Amita  , Sinha Kislay K. , Chaudhuri Saumya Ray , Garg Pallavi , Ramamurthy Thandavarayan , Nair Gopinath Balakrish , Ghosh Amit
Published: October 2002

Molecular mechanisms of multidrug resistance in
Vibrio cholerae
belonging to non-O1, non-O139 serogroups isolated during 1997 to 1998 in Calcutta, India, were investigated. Out of the 94 strains examined, 22 strains were found to have class I integrons. The gene cassettes identified were
dfrA1
,
dfrA15
,
dfrA5
, and
dfrA12
for trimethoprim;
aac
(
6
′)
-Ib
for amikacin and tobramycin;
aadA1
and
aadA2
for streptomycin and spectinomycin; and
ereA2
for erythromycin resistance. To our knowledge, this is the first report of the presence of
dfrA5
,
dfrA12
,
aac
(
6
′)
-Ib,
and
ereA2
cassettes in class I integrons of
V. cholerae
. Forty-three of 94 strains also had plasmids, and out of these, 14 contained both class I integrons and plasmids. Pulsed-field gel electrophoresis followed by Southern hybridization revealed that in the 14 plasmid-bearing strains, class I integrons resided either on chromosomes, on plasmids, or on both. Our results indicated that besides class I integrons and plasmids, a conjugative transposon element, SXT, possibly contributed to the multiple antibiotic resistance.
Predicting Evolution by In Vitro Evolution Requires Determining Evolutionary Pathways
Authors: Hall Barry G.
Published: October 2002

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Inducible Promoter-Repressor System from the
Lactobacillus casei
Phage φFSW
Authors: Binishofer Bernhard , Moll Isabella , Henrich Bernhard , Bläsi Udo
Published: October 2002

With the aim to extend the presently available inducible gene expression systems for lactobacilli, we have isolated a thermoinducible promoter-repressor cassette from the temperate
Lactobacillus casei
phage φFSW-TI in
Escherichia coli
. The φFSW-TI promoter fragment was abutted to the plasmid-borne promoterless β-glucuronidase (
gusA
) reporter gene and shown to direct its transcription in
L. casei
. In addition, the functionality of the promoter-repressor system was verified in the
L. casei
φFSW-TI lysogen by showing that the
gusA
reporter gene, controlled by the isolated φFSW-TI promoter, was repressed at 28°C and expressed at 42°C. Moreover, a homology search revealed that the C terminus of the isolated φFSW repressor shows a high similarity to the small
mutS
-related domain of the MutS2 protein family that is unprecedented for phage-encoded repressor proteins.
Inhibitory Effects of High Pressure and Heat on
Alicyclobacillus acidoterrestris
Spores in Apple Juice
Authors: Lee Sun-Young , Dougherty Richard H. , Kang Dong-Hyun
Published: October 2002

The effectiveness of combined high pressure and heat treatment for reducing spore levels of
Alicyclobacillus acidoterrestris
, a thermoacidophilic spore-forming bacterium, in commercial pasteurized apple juice was investigated. Spores suspended in apple juice were successfully destroyed by combining high pressure with a mild or high temperature (45, 71, or 90°C).
Cryptococcal Glucuronoxylomannan Inhibits Adhesion of Neutrophils to Stimulated Endothelium In Vitro by Affecting Both Neutrophils and Endothelial Cells
Authors: Ellerbroek Pauline M. , Hoepelman Andy I. M. , Wolbers Floor , Zwaginga Jaap Jan , Coenjaerts Frank E. J.
Published: October 2002

Cryptococcal infections are often characterized by a paucity of leukocytes in the infected tissues. Previous research has shown that the capsular polysaccharide glucuronoxylomannan (GXM) inhibits leukocyte migration. In this study we investigated whether the capsular polysaccharide GXM affects the migration of neutrophils (polymorphonuclear leukocytes [PMN]) through the endothelium by interfering with adhesion in a static adhesion model. Pretreatment of PMN with GXM inhibited PMN adhesion to tumor necrosis factor alpha (TNF-α)-stimulated endothelium up to 44%. Treatment of TNF-α-stimulated endothelium with GXM led to a 27% decrease in PMN adhesion. GXM treatment of both PMN and endothelium did not have an additive inhibitory effect. We demonstrated that GXM-induced L-selectin shedding does not play an important role in the detected inhibition of adhesion. L-selectin was still present on PMN in sufficient amounts after GXM treatment, since it could be further inhibited by blocking antibodies. Furthermore, blocking of GXM-related L-selectin shedding did not abolish the GXM-related inhibition of adhesion. GXM most likely exerts its effect on PMN by interfering with E-selectin-mediated binding. The use of blocking monoclonal antibodies against E-selectin, which was shown to decrease adhesion in the absence of GXM, did not cause additive inhibition of PMN adhesion after GXM pretreatment. The use of blocking antibodies also demonstrated that the inhibiting effect found after GXM treatment of endothelium probably involves interference with both intercellular adhesion molecule-1 and E-selectin binding.
Decreased Electroporation Efficiency in
Borrelia burgdorferi
Containing Linear Plasmids lp25 and lp56: Impact on Transformation of Infectious
B. burgdorferi
Authors: Lawrenz Matthew B. , Kawabata Hiroki , Purser Joye E. , Norris Steven J.
Published: October 2002

The presence of the linear plasmids lp25 and lp56 of
Borrelia burgdorferi
B31 was found to dramatically decrease the rate of transformation by electroporation with the shuttle vector pBSV2, an autonomously replicating plasmid that confers kanamycin resistance (P. E. Stewart, R. Thalken, J. L. Bono, and P. Rosa, Mol. Microbiol. 39:714-721, 2001).
B. burgdorferi
B31 clones had transformation efficiencies that were either low, intermediate, or high, and this phenotype correlated with the presence or absence of lp25 and lp56. Under the conditions utilized in this study, no transformants were detected in clones that contained both lp25 and lp56; the few kanamycin-resistant colonies isolated did not contain pBSV2, indicating that the resistance was due to mutation. Intermediate electroporation rates (10 to 200 colonies per μg of DNA) were obtained with B31 clones that were either lp25
−
and lp56
+
or lp25
+
and lp56
−
. Clones in this group that initially contained lp25 lacked this plasmid in pBSV2 transformants, a finding consistent with selective transformation of lp25
−
variants. High transformation rates (>1,000 colonies per μg of DNA) occurred in clones that lacked both lp25 and lp56. Sequence analysis indicated that lp25 and lp56 contain genes that may encode restriction and/or modification systems that could result in the low transformation rates obtained with strains containing these plasmids. The previously reported correlation between lp25 and infectivity in mice, coupled with the barrier lp25 presents to transformation, may explain the difficulty in obtaining virulent transformants of
B. burgdorferi
.