bluuebunny/crossref_metadata_2025 huggingface dataset:
Abiogenic Methane Formation and Isotopic Fractionation Under Hydrothermal Conditions
Authors: Horita Juske , Berndt Michael E.
Published: July 2002

Recently, methane (CH
4
) of possible abiogenic origin has been reported from many localities within Earth's crust. However, little is known about the mechanisms of abiogenic methane formation, or about isotopic fractionation during such processes. Here, a hydrothermally formed nickel-iron alloy was shown to catalyze the otherwise prohibitively slow formation of abiogenic CH
4
from dissolved bicarbonate (HCO
3
−
) under hydrothermal conditions. Isotopic fractionation by the catalyst resulted in δ
13
C values of the CH
4
formed that are as low as those typically observed for microbial methane, with similarly high CH
4
/(C
2
H
6
+ C
3
H
8
) ratios. These results, combined with the increasing recognition of nickel-iron alloy occurrence in oceanic crusts, suggest that abiogenic methane may be more widespread than previously thought.
Optical Measurements of Invasive Forces Exerted by Appressoria of a Plant Pathogenic Fungus
Authors: Bechinger Clemens , Giebel Karl-Friedrich , Schnell Martin , Leiderer Paul , Deising Holger B. , Bastmeyer Martin
Published: July 2002

Many plant pathogenic fungi, such as the cereal pathogen
Colletotrichum graminicola
, differentiate highly specialized infection structures called appressoria, which send a penetration peg into the underlying plant cell. Appressoria have been shown to generate enormous turgor pressure, but direct evidence for mechanical infection of plants by fungi is lacking. A microscopic method was developed that uses elastic optical waveguides to visualize and measure forces locally exerted by single appressoria. By this method, the force exerted by appressoria of
C. graminicola
was found to be about 17 micronewtons.
Control of the DNA Damage Checkpoint by Chk1 and Rad53 Protein Kinases Through Distinct Mechanisms
Authors: Sanchez Yolanda , Bachant Jeff , Wang Hong , Hu Fenghua , Liu Dou , Tetzlaff Michael , Elledge Stephen J.
Published: July 2002

In response to DNA damage, cells activate checkpoint pathways that prevent cell cycle progression. In fission yeast and mammals, mitotic arrest in response to DNA damage requires inhibitory Cdk phosphorylation regulated by Chk1. This study indicates that Chk1 is required for function of the DNA damage checkpoint in
Saccharomyces cerevisiae
but acts through a distinct mechanism maintaining the abundance of Pds1, an anaphase inhibitor. Unlike other checkpoint mutants,
chk1
mutants were only mildly sensitive to DNA damage, indicating that checkpoint functions besides cell cycle arrest influence damage sensitivity. Another kinase, Rad53, was required to both maintain active cyclin-dependent kinase 1, Cdk1(Cdc28), and prevent anaphase entry after checkpoint activation. Evidence suggests that Rad53 exerts its role in checkpoint control through regulation of the Polo kinase Cdc5. These results support a model in which Chk1 and Rad53 function in parallel through Pds1 and Cdc5, respectively, to prevent anaphase entry and mitotic exit after DNA damage. This model provides a possible explanation for the role of Cdc5 in DNA damage checkpoint adaptation.
Eight Centuries of North Atlantic Ocean Atmosphere Variability
Authors: Black David E. , Peterson Larry C. , Overpeck Jonathan T. , Kaplan Alexey , Evans Michael N. , Kashgarian Michaele
Published: July 2002

Climate in the tropical North Atlantic is controlled largely by variations in the strength of the trade winds, the position of the Intertropical Convergence Zone, and sea surface temperatures. A high-resolution study of Caribbean sediments provides a subdecadally resolved record of tropical upwelling and trade wind variability spanning the past 825 years. These results confirm the importance of a decadal (12- to 13-year) mode of Atlantic variability believed to be driven by coupled tropical ocean-atmosphere dynamics. Although a well-defined interdecadal mode of variability does not appear to be characteristic of the tropical Atlantic, there is evidence that century-scale variability is substantial. The tropical Atlantic may also have been involved in a major shift in Northern Hemisphere climate variability that took place about 700 years ago.
Nanotube Nanotweezers
Authors: Kim Philip , Lieber Charles M.
Published: July 2002

Nanoscale electromechanical systems—nanotweezers—based on carbon nanotubes have been developed for manipulation and interrogation of nanostructures. Electrically conducting and mechanically robust carbon nanotubes were attached to independent electrodes fabricated on pulled glass micropipettes. Voltages applied to the electrodes closed and opened the free ends of the nanotubes, and this electromechanical response was simulated quantitatively using known nanotweezer structure and nanotube properties. The mechanical capabilities of the nanotweezers were demonstrated by grabbing and manipulating submicron clusters and nanowires. The conducting nanotube arms of the tweezers were also used for measuring the electrical properties of silicon carbide nanoclusters and gallium arsenide nanowires.
Mediation by a CREB Family Transcription Factor of NGF-Dependent Survival of Sympathetic Neurons
Authors: Riccio Antonella , Ahn Sohyun , Davenport Christopher M. , Blendy Julie A. , Ginty David D.
Published: July 2002

Nerve growth factor (NGF) and other neurotrophins support survival of neurons through processes that are incompletely understood. The transcription factor CREB is a critical mediator of NGF-dependent gene expression, but whether CREB family transcription factors regulate expression of genes that contribute to NGF-dependent survival of sympathetic neurons is unknown. CREB-mediated gene expression was both necessary for NGF-dependent survival and sufficient on its own to promote survival of sympathetic neurons. Moreover, expression of Bcl-2 was activated by NGF and other neurotrophins by a CREB-dependent transcriptional mechanism. Overexpression of Bcl-2 reduced the death-promoting effects of CREB inhibition. Together, these data support a model in which neurotrophins promote survival of neurons, in part through a mechanism involving CREB family transcription factor–dependent expression of genes encoding prosurvival factors.
Inattentional Blindness Versus Inattentional Amnesia for Fixated But Ignored Words
Authors: Rees Geraint , Russell Charlotte , Frith Christopher D. , Driver Jon
Published: July 2002

People often are unable to report the content of ignored information, but it is unknown whether this reflects a complete failure to perceive it (inattentional blindness) or merely that it is rapidly forgotten (inattentional amnesia). Here functional imaging is used to address this issue by measuring brain activity for unattended words. When attention is fully engaged with other material, the brain no longer differentiates between meaningful words and random letters, even when they are looked at directly. These results demonstrate true inattentional blindness for words and show that visual recognition wholly depends on attention even for highly familiar and meaningful stimuli at the center of gaze.
Cross-Species Interactions Between Malaria Parasites in Humans
Authors: Bruce Marian C. , Donnelly Christl A. , Alpers Michael P. , Galinski Mary R. , Barnwell John W. , Walliker David , Day Karen P.
Published: July 2002

The dynamics of multiple
Plasmodium
infections in asymptomatic children living under intense malaria transmission pressure provide evidence for a density-dependent regulation that transcends species as well as genotype. This regulation, in combination with species- and genotype-specific immune responses, results in nonindependent, sequential episodes of infection with each species.
Facile Detection of Mitochondrial DNA Mutations in Tumors and Bodily Fluids
Authors: Fliss Makiko S. , Usadel Henning , Caballero Otávia L. , Wu Li , Buta Martin R. , Eleff Scott M. , Jen Jin , Sidransky David
Published: July 2002

Examination of human bladder, head and neck, and lung primary tumors revealed a high frequency of mitochondrial DNA (mtDNA) mutations. The majority of these somatic mutations were homoplasmic in nature, indicating that the mutant mtDNA became dominant in tumor cells. The mutated mtDNA was readily detectable in paired bodily fluids from each type of cancer and was 19 to 220 times as abundant as mutated nuclear p53 DNA. By virtue of their clonal nature and high copy number, mitochondrial mutations may provide a powerful molecular marker for noninvasive detection of cancer.
Splitting the Chromosome: Cutting the Ties That Bind Sister Chromatids
Authors: Nasmyth Kim , Peters Jan-Michael , Uhlmann Frank
Published: July 2002

In eukaryotic cells, sister DNA molecules remain physically connected from their production at S phase until their separation during anaphase. This cohesion is essential for the separation of sister chromatids to opposite poles of the cell at mitosis. It also permits chromosome segregation to take place long after duplication has been completed. Recent work has identified a multisubunit complex called cohesin that is essential for connecting sisters. Proteolytic cleavage of one of cohesin's subunits may trigger sister separation at the onset of anaphase.
Prokaryotic Regulation of Epithelial Responses by Inhibition of IκB-α Ubiquitination
Authors: Neish Andrew S. , Gewirtz Andrew T. , Zeng Hui , Young Andrew N. , Hobert Michael E. , Karmali Vinit , Rao Anjali S. , Madara James L.
Published: July 2002

Epithelia of the vertebrate intestinal tract characteristically maintain an inflammatory hyporesponsiveness toward the lumenal prokaryotic microflora. We report the identification of enteric organisms (nonvirulent
Salmonella
strains) whose direct interaction with model human epithelia attenuate synthesis of inflammatory effector molecules elicited by diverse proinflammatory stimuli. This immunosuppressive effect involves inhibition of the inhibitor κB/nuclear factor κB (IκB/NF-κB) pathway by blockade of IκB-α degradation, which prevents subsequent nuclear translocation of active NF-κB dimer. Although phosphorylation of IκB-α occurs, subsequent polyubiquitination necessary for regulated IκB-α degradation is completely abrogated. These data suggest that prokaryotic determinants could be responsible for the unique tolerance of the gastrointestinal mucosa to proinflammatory stimuli.
Integration of Multiple Signals Through Cooperative Regulation of the N-WASP-Arp2/3 Complex
Authors: Prehoda Kenneth E. , Scott Jessica A. , Mullins R. Dyche , Lim Wendell A.
Published: July 2002

The protein N-WASP [a homolog to the Wiskott-Aldrich syndrome protein (WASP)] regulates actin polymerization by stimulating the actin-nucleating activity of the actin-related protein 2/3 (Arp2/3) complex. N-WASP is tightly regulated by multiple signals: Only costimulation by Cdc42 and phosphatidylinositol (4,5)-bisphosphate (PIP
2
) yields potent polymerization. We found that regulation requires N-WASP's constitutively active output domain (VCA) and two regulatory domains: a Cdc42-binding domain and a previously undescribed PIP
2
-binding domain. In the absence of stimuli, the regulatory modules together hold the VCA-Arp2/3 complex in an inactive “closed” conformation. In this state, both the Cdc42- and PIP
2
-binding sites are masked. Binding of either input destabilizes the closed state and enhances binding of the other input. This cooperative activation mechanism shows how combinations of simple binding domains can be used to integrate and amplify coincident signals.
Genes, Genetics, and Epigenetics: A Correspondence
Authors: Wu C.-t. , Morris J. R.
Published: July 2002

Over the past months, as this special issue took shape, the Editors of
Science
have monitored an exchange of seven letters initiated by three queries from M. Bacon. These queries concern the popular definitions of “genes,” “genetics,” and “epigenetics.” Below, we reprint excerpts from these letters, referring interested readers to
www.sciencemag.org/cgi/content/full/293/5532/1103/DC1
for the complete text and additional references.
Activities of Clinafloxacin, Gatifloxacin, Gemifloxacin, and Trovafloxacin against Recent Clinical Isolates of Levofloxacin-Resistant
Streptococcus pneumoniae
Authors: Jorgensen J. H. , Weigel L. M. , Swenson J. M. , Whitney C. G. , Ferraro M. J. , Tenover F. C.
Published: July 2002

The activities of two investigational fluoroquinolones and three fluoroquinolones that are currently marketed were determined for 182 clinical isolates of
Streptococcus pneumoniae
. The collection included 57 pneumococcal isolates resistant to levofloxacin (MIC ≥ 8 μg/ml) recovered from patients in North America and Europe. All isolates were tested with clinafloxacin, gatifloxacin, gemifloxacin, levofloxacin, and trovafloxacin by the National Committee for Clinical Laboratory Standards broth microdilution and disk diffusion susceptibility test methods. Gemifloxacin demonstrated the greatest activity on a per gram basis, followed by clinafloxacin, trovafloxacin, gatifloxacin, and levofloxacin. Scatterplots of the MICs and disk diffusion zone sizes revealed a well-defined separation of levofloxacin-resistant and -susceptible strains when the isolates were tested against clinafloxacin and gatifloxacin. DNA sequence analyses of the quinolone resistance-determining regions of
gyrA
,
gyrB
,
parC
, and
parE
from 21 of the levofloxacin-resistant strains identified eight different patterns of amino acid changes. Mutations among the four loci had the least effect on the MICs of gemifloxacin and clinafloxacin, while the MICs of gatifloxacin and trovafloxacin increased by up to six doubling dilutions. These data indicate that the newer fluoroquinolones have greater activities than levofloxacin against pneumococci with mutations in the DNA gyrase or topoisomerase IV genes. Depending upon pharmacokinetics and safety, the greater potency of these agents could provide improved clinical efficacy against levofloxacin-resistant pneumococcal strains.
In Vitro Antimicrobial Susceptibility Testing of Bacterial Enteropathogens Causing Traveler's Diarrhea in Four Geographic Regions
Authors: Gomi Harumi , Jiang Zhi-Dong , Adachi Javier A. , Ashley David , Lowe Brett , Verenkar Mangala P. , Steffen Robert , DuPont Herbert L.
Published: July 2002

The emergence of resistant enteropathogens has been reported worldwide. Few data are available on the contemporary in vitro activities of commonly used antimicrobial agents against enteropathogens causing traveler's diarrhea (TD). The susceptibility patterns of antimicrobial agents currently available or under evaluation against pathogens causing TD in four different areas of the world were evaluated. Pathogens were identified in stool samples from U.S., Canadian, or European adults (18 years of age or older) with TD during 1997, visiting India, Mexico, Jamaica, or Kenya. MICs of 11different antimicrobials were determined against 284 bacterial enteropathogens by the agar dilution method. Ciprofloxacin, levofloxacin, ceftriaxone, and azithromycin were highly active in vitro against the enteropathogens, while traditional antimicrobials such as ampicillin, trimethoprim, and trimethoprim/sulfamethoxazole showed high levels and high frequencies of resistance. Rifaximin, a promising and poorly absorbable drug, had an MIC at which 90% of the strains tested were inhibited of 32 μg/ml, 250 times lower than the concentration of this drug in the stools. Amdinocillin, nalidixic acid, and doxycycline showed moderate activity. Fluoroquinolones are still the drugs of choice for TD in most regions of the world, although our study has a limitation due to the lack of
Escherichia coli
samples from Kenya and possible bias in selection of the patients for evaluation. Azithromycin and rifaximin should be considered as promising new agents. The widespread in vitro resistance of the traditional antimicrobial agents reported since the 1980s and the new finding of resistance to fluoroquinolones in Southeast Asia are the main reasons for monitoring carefully the antimicrobial susceptibility patterns worldwide and for developing and evaluating new antimicrobial agents for the treatment of TD.
Effect of Endotoxin on P-Glycoprotein-Mediated Biliary and Renal Excretion of Rhodamine-123 in Rats
Authors: Ando Hideyuki , Nishio Yuki , Ito Katsuki , Nakao Akimasa , Wang Li , Zhao Ying Lan , Kitaichi Kiyoyuki , Takagi Kenzo , Hasegawa Takaaki
Published: July 2002

The effects of
Klebsiella pneumoniae
endotoxin on the biliary excretion and renal handling of rhodamine-123 were investigated in rats at different times after intraperitoneal injection (1 mg/kg of body weight). The typical substrates for P glycoprotein, i.e., cyclosporine, colchicine, and erythromycin, inhibited the biliary clearance of rhodamine-123, whereas a substrate for organic cation transporter, cimetidine, did not inhibit clearance, suggesting that rhodamine-123 is transported mainly by P glycoprotein. The biliary, renal, and tubular secretory clearances of rhodamine-123 and the glomerular filtration rate significantly decreased 6 h after injection of endotoxin but returned to control levels by 24 h. These results suggest that endotoxin-induced decreases in P-glycoprotein-mediated biliary excretion and renal handling of rhodamine-123 were probably due to impairment of P-glycoprotein-mediated transport ability. Pretreatment with pentoxifylline (50 mg/kg) significantly inhibited endotoxin-induced increases in tumor necrosis factor alpha (TNF-α) levels in plasma, which ameliorated the endotoxin-induced reduction of the biliary excretion of rhodamine-123. It is likely that endotoxin-induced impairment of the transport of rhodamine-123 is caused, in part, by overproduction of TNF-α. The effect of endotoxin on the expression of P-glycoprotein mRNA in liver and kidneys of rats was investigated by using a reverse transcriptase PCR. The expression of Mdr1a mRNA in both liver and kidney decreased 6 h after endotoxin injection and returned to control levels after 24 h, whereas the expression of Mdr1b mRNA in liver increased at both times and that in kidney decreased at 24 h. These findings suggest that
K. pneumoniae
endotoxin dramatically decreases P-glycoprotein-mediated biliary and renal excretion of rhodamine-123 probably by decreasing the expression of Mdr1a, which is likely due to increased plasma TNF-α levels.
Effects of Renal Function on Pharmacokinetics of Recombinant Human Granulocyte Colony-Stimulating Factor in Lung Cancer Patients
Authors: Fukuda Masaaki , Oka Mikio , Ishida Yoshimasa , Kinoshita Haruki , Terashi Kenji , Fukuda Minoru , Kawabata Shigeru , Kinoshita Akitoshi , Soda Hiroshi , Kohno Shigeru
Published: July 2002

Animal studies suggest that the kidney is involved in the elimination of recombinant human granulocyte colony-stimulating factor (rhG-CSF), which is used for patients with neutropenia during cancer chemotherapy. Since anticancer drugs induce nephrotoxicity, it is important to clarify the role of the kidney in the pharmacokinetics of rhG-CSF in cancer patients. Our study was designed to evaluate the relationship between the pharmacokinetics of rhG-CSF and renal function in lung cancer patients compared to the absolute neutrophil count (ANC). The pharmacokinetic studies were conducted with 25 lung cancer patients. Following chemotherapy using platinum-based compounds, a bolus 5 μg of rhG-CSF/kg of body weight was intravenously injected from the first day of leukopenia or neutropenia. Pharmacokinetic parameters were estimated by fitting the concentration in serum-time data to a two-compartment model according to the population pharmacokinetics and the Bayesian method. Creatinine clearance (CLCR) was predicted by the Cockcroft-Gault formula. rhG-CSF clearance (CLG-CSF) correlated significantly with the ANC (r= 0.613;P< 0.001) and CLCR(r= 0.632;P< 0.001). Multiple linear regression analysis showed that the combination of the ANC and CLCRaccounted for 57.4% of the variation of CLG-CSF. In patients with an ANC of <1,000/μl, CLCRaccounted for 72.9% of the variation of CLG-CSF(P< 0.001). Our findings suggest that renal function and neutrophil counts correlate with CLG-CSFand that the role of renal function in eliminating rhG-CSF is important in lung cancer patients with neutropenia.
Genes Encoding TEM-4, SHV-2, and CTX-M-10 Extended-Spectrum β-Lactamases Are Carried by MultipleKlebsiella pneumoniaeClones in a Single Hospital (Madrid, 1989 to 2000)
Authors: Coque Teresa M. , Oliver Antonio , Pérez-Díaz José Claudio , Baquero Fernando , Cantón Rafael
Published: July 2002

Over a 12-year period (1989 to 2000), 159Klebsiella pneumoniaeisolates harboring extended-spectrum β-lactamases (ESBLs) (4.8% of the total number ofK. pneumoniaeisolates obtained) were recovered from 58 patients, who were mainly hospitalized in intensive care and surgery units. For 62 representative isolates from 58 patients, 31 clonal types harboring TEM-4 (n= 5), SHV-2 (n= 7), SHV2a (n= 4), SHV-5 (n= 1), CTX-M-10 (n= 13), or CTX-M-9 (n= 1) β-lactamases were identified by pulsed-field gel electrophoresis. This is the first report to document the presence of the CTX-M-10 or the CTX-M-9 β-lactamase inK. pneumoniae. These β-lactamases were previously identified inEscherichia coliisolates from Spain. Only two of fiveK. pneumoniaeTEM-4 clones caused more than a single case of infection, with one of them spreading for 9 months. A single plasmid was detected among these TEM-4 clones. Only two of sevenK. pneumoniaeclones containing SHV-2 and three of four strains harboring SHV-2a were detected in more than one case of infection. Plasmids encoding SHV-2 or SHV-2a were unrelated. Four of 13K. pneumoniaeCTX-M-10 clones were found in more than one patient, with two of them recovered 2 and 5 years apart. As in the case of the SHV-2 isolates, we were unable to document a common transmissible genetic element that could explain the polyclonal structure of our isolates. Nevertheless, the spread of a single gene may be suggested by the presence of a conserved set of noncoding polymorphisms in the sequences. Most ESBL-producingK. pneumoniaeclones were ephemeral, being poorly selected and maintained in the hospital setting, but the genes encoding ESBL persisted successfully over the years that the strains were recovered, probably as a minority gene population in the hospital metagenome.
Epigallocatechin Gallate Synergistically Enhances the Activity of Carbapenems against Methicillin-Resistant
Staphylococcus aureus
Authors: Hu Zhi-Qing , Zhao Wei-Hua , Asano Nozomi , Yoda Yoshiyuki , Hara Yukihiko , Shimamura Tadakatsu
Published: July 2002

Combinations of carbapenems and epigallocatechin gallate (EGCg; a main constituent of tea catechins) showed potent synergy against 24 clinical isolates of methicillin-resistant
Staphylococcus aureus
(MRSA). MICs of imipenem in the presence of EGCg at 3.125, 6.25, 12.5, and 25 μg/ml, were restored to the susceptible breakpoint (≤4 μg/ml) for 8, 38, 46, and 75% of the MRSA isolates, respectively. Similar results were also observed for combinations of panipenem or meropenem and EGCg. Therefore, the combinations may be worthy of further evaluation in vivo against MRSA infection.
Dose Range Study of Pharmacokinetics, Safety, and Preliminary Antiviral Activity of Emtricitabine in Adults with Hepatitis B Virus Infection
Authors: Gish R. G. , Leung N. W. Y. , Wright T. L. , Trinh Huy , Lang W. , Kessler H. A. , Fang L. , Wang L. H. , Delehanty J. , Rigney A. , Mondou E. , Snow A. , Rousseau F.
Published: July 2002

A multicenter, open-label study was performed to evaluate the safety, anti-hepatitis B virus (anti-HBV) activity, and pharmacokinetics of emtricitabine therapy administered once daily for 8 weeks to patients infected with HBV. Clinical and virologic evaluations were completed at the baseline; at 7, 14, 28, 42, and 56 days during treatment; and at 24, 48, and 28 days posttreatment. Forty-nine patients were enrolled in five dose cohorts (doses of 25, 50, 100, 200, and 300 mg, all of which were administered once daily [q.d.]). Peak plasma emtricitabine concentrations occurred within 1.5 h following dosing. Plasma emtricitabine concentrations (maximum concentrations of drug in plasma and areas under the concentration-time curves) increased nearly dose proportionally over the 25- to 300-mg dose range, with relatively small intersubject variabilities. The plasma half-life of emtricitabine ranged from 6 to 9 h. HBV DNA levels were measured by the Digene HBV Hybrid Capture II assay. Viral suppression (reduction in log
10
serum HBV DNA levels) occurred in all dose cohorts. All doses demonstrated potent and rapid antiviral activities, with a trend toward a greater suppression with daily doses of 100 mg or greater. At 2 months, the median change in the serum HBV DNA level from the baseline level ranged from −1.7 log
10
for the 25-mg dose administered q.d. to −3.3 log
10
for the 300 mg dose administered q.d. Emtricitabine was well tolerated over the 2-month dosing period. These results support further clinical development of emtricitabine for the treatment of chronic hepatitis B infection.
Correlations between Morphological, Molecular Biological, and Physiological Characteristics in Clinical and Nonclinical Isolates of
Acanthamoeba
spp
Authors: Walochnik Julia , Obwaller Andreas , Aspöck Horst
Published: July 2002

Eleven
Acanthamoeba
isolates, obtained from
Acanthamoeba
keratitis patients, from contact lens cases of non-
Acanthamoeba
keratitis patients, from asymptomatic individuals, from necrotic tissue, and from tap water and two reference strains were investigated by morphological, molecular biological, and physiological means in order to discriminate clinically relevant and nonrelevant isolates. All clinically relevant isolates showed
Acanthamoeba
sp. group II morphology. 18S ribosomal DNA sequencing revealed sequence type T4 to be the most prevalent group among the isolates and also the group recruiting most of the pathogenic strains. Interestingly, within T4 the strains of no clinical relevance clustered together. Moreover, physiological properties appeared to be highly consistent with initial pathogenicity and with sequence clustering. Altogether, the results of our study indicate a correlation between the phylogenetic relationship and pathogenicity.
Genetic Variation in
Fusarium
Section
Liseola
from No-Till Maize in Argentina
Authors: Chulze S. N. , Ramirez M. L. , Torres A. , Leslie J. F.
Published: July 2002

Strains of
Fusarium
species belonging to section
Liseola
cause stalk and ear rot of maize and produce important mycotoxins, such as fumonisins. We isolated two species,
Fusarium verticillioides
(
Gibberella fujikuroi
mating population A) and
Fusarium proliferatum
(
G. fujikuroi
mating population D) from maize cultivated under no-till conditions at five locations in the Córdoba province of Argentina. We determined the effective population number for mating population A (

N
e

) and found that the

N
e

for mating type was 89% of the count (total population) and that the

N
e

for male or hermaphrodite status was 36%. Thus, the number of strains that can function as the female parent limits

N
e

, and sexual reproduction needs to occur only once every 54 to 220 asexual generations to maintain this level of sexual fertility. Our results indicate that the fungal populations isolated from no-till maize are similar to those recovered from maize managed with conventional tillage. We placed 36 strains from mating population A into 28 vegetative compatibility groups (VCGs). Of the 13 strains belonging to five multimember VCGs, only 2 isolates belonging to one VCG were clones based on amplified fragment length polymorphism (AFLP) fingerprints. Members of the other four multimember VCGs had an average similarity index of 0.89, and members of one VCG were no more closely related to other members of the same VCG than they were to other members of the population as a whole. This finding suggests that the common assumption that strains in the same VCG are either clonal or very closely related needs to be examined in more detail. The variability observed with AFLPs and VCGs suggests that sexual reproduction may occur more frequently than estimated by

N
e

.
Purification and Molecular Characterization of a Tripeptidase (PepT) from
Lactobacillus helveticus
Authors: Savijoki Kirsi , Palva Airi
Published: July 2002

A tripeptidase (PepT) from a thermophilic dairy starter strain of
Lactobacillus helveticus
was purified by four chromatographic steps. PepT appeared to be a trimeric metallopeptidase with a molecular mass of 150 kDa. PepT exhibited maximum activity against hydrophobic tripeptides, with the highest activity for Met-Gly-Gly (

K
m

, 2.6 mM;
V
max
, 80.2 μmol · min
−1
· μg
−1
). Some of the hydrophobic dipeptides were slowly hydrolyzed, distinguishing the
Lactobacillus
PepT from its counterpart in mesophilic
Lactococcus lactis
. No activity against tetrapeptides or amino acid
p
-nitroanilide derivatives was observed. The
pepT
gene and its flanking regions were isolated by PCR and sequenced by cyclic sequencing. The sequence analyses revealed open reading frames (ORFs) 816 bp (ORF1) and 1,239 bp (ORF2) long. ORF2 encoded a 47-kDa PepT protein which exhibited 53% identity with the PepT from
L. lactis
. The mRNA analyses indicated that
pepT
conforms a novel operon structure with an ORF1 located upstream. Several putative −35/−10 regions preceded the operon, but only one transcription start site located downstream of the first putative −10 region was identified. An inverted repeat structure with Δ
G
of −64.8 kJ/mol was found downstream of the PepT-encoding region.
Detection of DNA Damage in Prokaryotes by Terminal Deoxyribonucleotide Transferase-Mediated dUTP Nick End Labeling
Authors: Rohwer Forest , Azam Farooq
Published: July 2002

Numerous agents can damage the DNA of prokaryotes in the environment (e.g., reactive oxygen species, irradiation, and secondary metabolites such as antibiotics, enzymes, starvation, etc.). The large number of potential DNA-damaging agents, as well as their diverse modes of action, precludes a simple test of DNA damage based on detection of nucleic acid breakdown products. In this study, free 3′-OH DNA ends, produced by either direct damage or excision DNA repair, were used to assess DNA damage. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) is a procedure in which 3′-OH DNA ends are enzymatically labeled with dUTP-fluorescein isothiocyanate using TdT. Cells labeled by this method can be detected using fluorescence microscopy or flow cytometry. TUNEL was used to measure hydrogen peroxide-induced DNA damage in the archaeon
Haloferax volcanii
and the bacterium
Escherichia coli
. DNA repair systems were implicated in the hydrogen peroxide-dependent generation of 3′-OH DNA ends by the finding that the protein synthesis inhibitors chloramphenicol and diphtheria toxin blocked TUNEL labeling of
E. coli
and
H. volcanii
, respectively. DNA damage induced by UV light and bacteriophage infection was also measured using TUNEL. This methodology should be useful in applications where DNA damage and repair are of interest, including mutant screening and monitoring of DNA damage in the environment.
Gene Cloning and Nucleotide Sequencing and Properties of a Cocaine Esterase fromRhodococcussp. Strain MB1
Authors: Bresler Matthew M. , Rosser Susan J. , Basran Amrik , Bruce Neil C.
Published: July 2002

A strain ofRhodococcusdesignated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plantErythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized byRhodococcussp. strain MB1. The structural gene encoding a cocaine esterase, designatedcocE, was cloned fromRhodococcussp. strain MB1 genomic libraries by screening recombinant strains ofRhodococcus erythropolisCW25 for growth on cocaine. The nucleotide sequence ofcocEcorresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. ThecocEcoding sequence was subcloned into the pCFX1 expression plasmid and expressed inEscherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with anMrof approximately 65,000. The apparentKmof the enzyme (mean ± standard deviation) for cocaine was measured as 1.33 ± 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.
Relationships between Microbial Community Structure and Hydrochemistry in a Landfill Leachate-Polluted Aquifer
Authors: Röling Wilfred F. M. , van Breukelen Boris M. , Braster Martin , Lin Bin , van Verseveld Henk W.
Published: July 2002

Knowledge about the relationship between microbial community structure and hydrogeochemistry (e.g., pollution, redox and degradation processes) in landfill leachate-polluted aquifers is required to develop tools for predicting and monitoring natural attenuation. In this study analyses of pollutant and redox chemistry were conducted in parallel with culture-independent profiling of microbial communities present in a well-defined aquifer (Banisveld, The Netherlands). Degradation of organic contaminants occurred under iron-reducing conditions in the plume of pollution, while upstream of the landfill and above the plume denitrification was the dominant redox process. Beneath the plume iron reduction occurred. Numerical comparison of 16S ribosomal DNA (rDNA)-based denaturing gradient gel electrophoresis (DGGE) profiles of
Bacteria
and
Archaea
in 29 groundwater samples revealed a clear difference between the microbial community structures inside and outside the contaminant plume. A similar relationship was not evident in sediment samples. DGGE data were supported by sequencing cloned 16S rDNA. Upstream of the landfill members of the β subclass of the class
Proteobacteria
(β-proteobacteria) dominated. This group was not encountered beneath the landfill, where gram-positive bacteria dominated. Further downstream the contribution of gram-positive bacteria to the clone library decreased, while the contribution of δ-proteobacteria strongly increased and β-proteobacteria reappeared. The β-proteobacteria (
Acidovorax
,
Rhodoferax
) differed considerably from those found upstream (
Gallionella
,
Azoarcus
). Direct comparisons of cloned 16S rDNA with bands in DGGE profiles revealed that the data from each analysis were comparable. A relationship was observed between the dominant redox processes and the bacteria identified. In the iron-reducing plume members of the family
Geobacteraceae
made a strong contribution to the microbial communities. Because the only known aromatic hydrocarbon-degrading, iron-reducing bacteria are
Geobacter
spp., their occurrence in landfill leachate-contaminated aquifers deserves more detailed consideration.
Identification by Subtractive Hybridization of Sequences Specific for
Salmonella enterica
Serovar Enteritidis
Authors: Agron Peter G. , Walker Richard L. , Kinde Hailu , Sawyer Sherilyn J. , Hayes Dawn C. , Wollard Jessica , Andersen Gary L.
Published: July 2002

Salmonella enterica
serovar Enteritidis, a major cause of food poisoning, can be transmitted to humans through intact chicken eggs when the contents have not been thoroughly cooked. Infection in chickens is asymptomatic; therefore, simple, sensitive, and specific detection methods are crucial for efforts to limit human exposure. Suppression subtractive hybridization was used to isolate DNA restriction fragments present in
Salmonella
serovar Enteritidis but absent in other bacteria found in poultry environments. Oligonucleotide primers to candidate regions were used in polymerase chain reactions to test 73 non-Enteritidis
S. enterica
isolates comprising 34 different serovars, including Dublin and Pullorum, two very close relatives of Enteritidis. A primer pair to one
Salmonella
difference fragment (termed Sdf I) clearly distinguished serovar Enteritidis from all other serovars tested, while two other primer pairs only identified a few non-Enteritidis strains. These primer pairs were also useful for the detection of a diverse collection of clinical and environmental
Salmonella
serovar Enteritidis isolates. In addition, five bacterial genera commonly found with
Salmonella
serovar Enteritidis were not detected. By treating total DNA with an exonuclease that degrades sheared chromosomal DNA but not intact circular plasmid DNA, it was shown that Sdf I is located on the chromosome. The Sdf I primers were used to screen a
Salmonella
serovar Enteritidis genomic library and a unique 4,060-bp region was defined. These results provide a basis for developing a rapid, sensitive, and highly specific detection system for
Salmonella
serovar Enteritidis and provide sequence information that may be relevant to the unique characteristics of this serovar.
Identification of Bacteriocin-Like Inhibitors from Rumen
Streptococcus
spp. and Isolation and Characterization of Bovicin 255
Authors: Whitford M. F. , McPherson M. A. , Forster R. J. , Teather R. M.
Published: July 2002

Streptococci obtained from rumen sources were tested for the production of antibacterial compounds using a deferred-antagonism plating assay. Of 35 isolates tested, 7 were identified that inhibited the growth of other streptococci. None of the inhibitory activity was due to bacteriophage. Three isolates, LRC0253, LRC0255, and LRC0476, were selected for further characterization. Analysis of 16S ribosomal DNA indicated that LRC0476 was a strain of
Streptococcus bovis
, while isolates LRC0253 and LRC0255 are likely strains of
Streptococcus gallolyticus
. The antibacterial compounds produced by these bacteria were protease sensitive, remained active in a pH range from 1 to 12, and did not lose activity after heating at 100°C for 15 min. The inhibitory peptide from strain LRC0255 was purified using pH-dependent adsorption and desorption to bacterial cells, followed by ammonium sulfate precipitation and reversed-phase chromatography and gel filtration. The peptide was 6 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An oligonucleotide probe based on the N-terminal sequence of the purified peptide was used to identify the gene encoding the inhibitory peptide. The antibacterial peptide has characteristics that are very similar to those described for class II bacteriocins of gram-positive bacteria.
Clostridium beijerinckii
and
Clostridium difficile
Detoxify Methylglyoxal by a Novel Mechanism Involving Glycerol Dehydrogenase
Authors: Liyanage Hemachandra , Kashket Shelby , Young Michael , Kashket Eva R.
Published: July 2002

In contrast to gram-negative bacteria, little is known about the mechanisms by which gram-positive bacteria degrade the toxic metabolic intermediate methylglyoxal (MG).
Clostridium beijerinckii
BR54, a Tn
1545
insertion mutant of the NCIMB 8052 strain, formed cultures that contained significantly more (free) MG than wild-type cultures. Moreover, BR54 was more sensitive to growth inhibition by added MG than the wild type, suggesting that it has a reduced ability to degrade MG. The single copy of Tn
1545
in this strain lies just downstream from
gldA
, encoding glycerol dehydrogenase. As a result of antisense RNA production, cell extracts of BR54 possess significantly less glycerol dehydrogenase activity than wild-type cell extracts (H. Liyanage, M. Young, and E. R. Kashket, J. Mol. Microbiol. Biotechnol. 2:87–93, 2000). Inactivation of
gldA
in both
C. beijerinckii
and
Clostridium difficile
gave rise to pinpoint colonies that could not be subcultured, indicating that glycerol dehydrogenase performs an essential function in both organisms. We propose that this role is detoxification of MG. To our knowledge, this is the first report of targeted gene disruption in the
C. difficile
chromosome.
Detection of Acute Bee Paralysis Virus and Black Queen Cell Virus from Honeybees by Reverse Transcriptase PCR
Authors: Benjeddou Mongi , Leat Neil , Allsopp Mike , Davison Sean
Published: July 2002

A reverse transcriptase PCR (RT-PCR) assay was developed for the detection of acute bee paralysis virus (ABPV) and black queen cell virus (BQCV), two honeybee viruses. Complete genome sequences were used to design unique PCR primers within a 1-kb region from the 3′ end of both genomes to amplify a fragment of 900 bp from ABPV and 700 bp from BQCV. The combined guanidinium thiocyanate and silica membrane method was used to extract total RNA from samples of healthy and laboratory-infected bee pupae. In a blind test, RT-PCR successfully identified the samples containing ABPV and BQCV. Sensitivities were approximately 1,600 genome equivalents of purified ABPV and 130 genome equivalents of BQCV.
Genetic Diversity of
Nostoc
Symbionts Endophytically Associated with Two Bryophyte Species
Authors: Costa José-Luis , Paulsrud Per , Rikkinen Jouko , Lindblad Peter
Published: July 2002

The diversity of the endophytic
Nostoc
symbionts of two thalloid bryophytes, the hornwort
Anthoceros fusiformis
and the liverwort
Blasia pusilla
, was examined using the tRNA
Leu
(UAA) intron sequence as a marker. The results confirmed that many different
Nostoc
strains are involved in both associations under natural conditions in the field. The level of
Nostoc
diversity within individual bryophyte thalli varied, but single DNA fragments were consistently amplified from individual symbiotic colonies. Some
Nostoc
strains were widespread and were detected from thalli collected from different field sites and different years. These findings indicate a moderate level of spatial and temporal continuity in bryophyte-
Nostoc
symbioses.
Surface Modifications Created by Using Engineered Hydrophobins
Authors: Scholtmeijer Karin , Janssen Meike I. , Gerssen Bertus , de Vocht Marcel L. , van Leeuwen Babs M. , van Kooten Theo G. , Wösten Han A. B. , Wessels Joseph G. H.
Published: July 2002

Hydrophobins are small (ca. 100 amino acids) secreted fungal proteins that are characterized by the presence of eight conserved cysteine residues and by a typical hydropathy pattern. Class I hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into highly insoluble amphipathic membranes, thereby changing the nature of surfaces. Hydrophobic surfaces become hydrophilic, while hydrophilic surfaces become hydrophobic. To see whether surface properties of assembled hydrophobins can be changed, 25
N
-terminal residues of the mature SC3 hydrophobin were deleted (TrSC3). In addition, the cell-binding domain of fibronectin (RGD) was fused to the N terminus of mature SC3 (RGD-SC3) and TrSC3 (RGD-TrSC3). Self-assembly and surface activity were not affected by these modifications. However, physiochemical properties at the hydrophilic side of the assembled hydrophobin did change. This was demonstrated by a change in wettability and by enhanced growth of fibroblasts on Teflon-coated with RGD-SC3, TrSC3, or RGD-TrSC3 compared to bare Teflon or Teflon coated with SC3. Thus, engineered hydrophobins can be used to functionalize surfaces.
Engineering of Phytase for Improved Activity at Low pH
Authors: Tomschy Andrea , Brugger Roland , Lehmann Martin , Svendsen Allan , Vogel Kurt , Kostrewa Dirk , Lassen Søren F. , Burger Dominique , Kronenberger Alexandra , van Loon Adolphus P. G. M. , Pasamontes Luis , Wyss Markus
Published: July 2002

For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles ofAspergillus fumigatusand consensus phytases. Decreasing the negative surface charge of theA. fumigatusQ27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH ofA. nigerNRRL 3135 phytase,A. nigerpH 2.5 acid phosphatase, andPeniophora lyciiphytase. Site-directed mutagenesis confirmed that, inA. fumigatuswild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues ofA. nigerphytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in bothA. fumigatusand consensus phytase backbones), as well as the S140Y D141G double mutation (inA. fumigatusphytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.
Ethyleneglycol-Bis-(β-Aminoethylether)Tetraacetate as a Blood Anticoagulant: Preservation of Antigen-Presenting Cell Function and Antigen-Specific Proliferative Response of Peripheral Blood Mononuclear Cells from Stored Blood
Authors: Kumar Priti , Satchidanandam Vijaya
Published: July 2002

We examined the progressive and irreversible loss of antigen-specific lymphoproliferative responses in peripheral blood mononuclear cells (PBMC) obtained from blood exposed for prolonged periods to EDTA as an anticoagulant. The responses of these lymphocytes to interleukin-2 or to concanavalin A were, however, unaffected. The observed loss was not due to depletion of metal ions by EDTA, since the addition of several divalent cations to whole blood during storage in EDTA or to lymphocytes from EDTA-stored blood during antigen stimulation in vitro did not alleviate the defect. Reconstitution of antigen-specific T-cell lines or Percoll-purified T cells with adherent antigen-presenting cells in antigen stimulation assays revealed that the presenting cells and not the effector T-cells were the targets of EDTA-mediated damage. The anticoagulant heparin helped to circumvent this problem. Surprisingly, EGTA, another metal ion chelator, could successfully replace EDTA, with a marginal loss in antigen-specific responses. Lymphoproliferative responses to antigens of Japanese encephalitis virus (JEV) andMycobacterium tuberculosiswere both significantly preserved in EGTA. JEV antigen-specific responses of PBMC obtained from the blood of convalescent JEV patients and stored in EGTA for as long as 24 h (n= 20) were comparable to those of fresh PBMC (n= 10), while PBMC from blood stored in EDTA (n= 17) for 16 h or longer failed to respond. We recommend that EGTA be used as the anticoagulant of choice for applications that require the lymphocyte proliferation assay, especially when on-site testing facilities are not available.
Competitive Enzyme-Linked Immunosorbent Assay for Detection of
Leptospira interrogans
Serovar pomona Antibodies in Bovine Sera
Authors: Surujballi Om , Mallory Maria
Published: July 2002

A competitive enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody (M898) was developed for detection of bovine antibodies to
Leptospira interrogans
serovar pomona. This assay was evaluated using field sera (
n
= 190) with serovar pomona microscopic agglutination test (MAT) titers of ≥100 as the positive population (group A); field sera (
n
= 1,445) which were negative in the MAT (1:100 dilution) for serovar pomona (group B); and sera (from a specific-pathogen-free cattle herd [
n
= 210]) which were negative in the MAT (1:100 dilution) for serovars canicola, copenhageni, grippotyphosa, hardjo, pomona, and sejroe (group C). At the cutoff point recommended by receiver operating characteristic (ROC) curve analysis of the combined ELISA results of serum groups A, B, and C, the sensitivity and specificity values were 93.7 and 96.3%, respectively. The value for the area under this ROC curve was 0.977, indicating a high level of accuracy for the ELISA. Similar results were obtained from the analysis of the combined results of serum groups A and B and from the analysis of the combined results of serum groups A and C.