bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Anthropogenic Climate Change in the World's Oceans
Authors: Barnett Tim P. , Pierce David W. , Schnur Reiner
Published: July 2002

Large-scale increases in the heat content of the world's oceans have been observed to occur over the last 45 years. The horizontal and temporal character of these changes has been closely replicated by the state-of-the-art Parallel Climate Model (PCM) forced by observed and estimated anthropogenic gases. Application of optimal detection methodology shows that the model-produced signals are indistinguishable from the observations at the 0.05 confidence level. Further, the chances of either the anthropogenic or observed signals being produced by the PCM as a result of natural, internal forcing alone are less than 5%. This suggests that the observed ocean heat-content changes are consistent with those expected from anthropogenic forcing, which broadens the basis for claims that an anthropogenic signal has been detected in the global climate system. Additionally, the requirement that modeled ocean heat uptakes match observations puts a strong, new constraint on anthropogenically forced climate models. It is unknown if the current generation of climate models, other than the PCM, meet this constraint.
Large Groundwater Strontium Flux to the Oceans from the Bengal Basin and the Marine Strontium Isotope Record
Authors: Basu Asish R. , Jacobsen Stein B. , Poreda Robert J. , Dowling Carolyn B. , Aggarwal Pradeep K.
Published: July 2002

Strontium concentration and isotopic data for subsurface flowing groundwaters of the Ganges-Brahmaputra (G-B) delta in the Bengal Basin demonstrate that this is a potentially significant source of strontium to the oceans, equal in magnitude to the dissolved strontium concentration carried to the oceans by the G-B river waters. The strontium concentrations of groundwaters are higher by a factor of about 10 than typical G-B river waters and they have similar
87
Sr/
86
Sr ratio to the river waters. These new data suggest that the present contribution of the G-B system to the rise in
87
Sr/
86
Sr ratio in seawater is higher by at least a factor of 2 to 5 than the average over the past 40 million years.
Phosphorylation-Dependent Ubiquitination of Cyclin E by the SCF
Fbw7
Ubiquitin Ligase
Authors: Koepp Deanna M. , Schaefer Laura K. , Ye Xin , Keyomarsi Khandan , Chu Claire , Harper J. Wade , Elledge Stephen J.
Published: July 2002

Cyclin E binds and activates the cyclin-dependent kinase Cdk2 and catalyzes the transition from the G
1
phase to the S phase of the cell cycle. The amount of cyclin E protein present in the cell is tightly controlled by ubiquitin-mediated proteolysis. Here we identify the ubiquitin ligase responsible for cyclin E ubiquitination as SCF
Fbw7
and demonstrate that it is functionally conserved in yeast, flies, and mammals. Fbw7 associates specifically with phosphorylated cyclin E, and SCF
Fbw7
catalyzes cyclin E ubiquitination in vitro. Depletion of Fbw7 leads to accumulation and stabilization of cyclin E in vivo in human and
Drosophila melanogaster
cells. Multiple F-box proteins contribute to cyclin E stability in yeast, suggesting an overlap in SCF E3 ligase specificity that allows combinatorial control of cyclin E degradation.
Identification of BIME as a Subunit of the Anaphase-Promoting Complex
Authors: Peters Jan-Michael , King Randall W. , Höög Christer , Kirschner Marc W.
Published: July 2002

The initiation of anaphase and exit from mitosis require the activation of a proteolytic system that ubiquitinates and degrades cyclin B. The regulated component of this system is a large ubiquitin ligase complex, termed the anaphase-promoting complex (APC) or cyclosome. Purified
Xenopus laevis
APC was found to be composed of eight major subunits, at least four of which became phosphorylated in mitosis. In addition to CDC27, CDC16, and CDC23, APC contained a homolog of
Aspergillus nidulans
BIME, a protein essential for anaphase. Because mutation of
bimE
can bypass the interphase arrest induced by either
nimA
mutation or unreplicated DNA, it appears that ubiquitination catalyzed by APC may also negatively regulate entry into mitosis.
Planet Within a Planet: Rotation of the Inner Core of Earth
Authors: Su Wei-jia , Dziewonski Adam M. , Jeanloz Raymond
Published: July 2002

The time dependence of the orientation of Earth's inner core relative to the mantle was determined using a recently discovered 10-degree tilt in the axis of symmetry of the inner core's seismic-velocity anisotropy. Two methods of analyzing travel-time variations for rays traversing the inner core, on the basis of 29 years of data from the International Seismological Centre (1964–1992), reveal that the inner core appears to rotate about 3 degrees per year faster than the mantle. An anomalous variation in inner-core orientation from 1969 to 1973 coincides in time with a sudden change (“jerk”) in the geomagnetic field.
Nematic Emulsions
Authors: Joanny J. F.
Published: July 2002

Emulsions and colloids are fundamentally interesting but also have technological importance in industrial applications. In his Perspective, Joanny [see also Poulin
et al
. (
p. 1770
)] discusses results reported in the same issue on a new kind of colloidal interaction observed when water droplets are dispersed in a liquid-crystal solvent. This liquid-crystal emulsion provides a way to understand the formation and stability of defects in complex fluids.
Bioinformatics: Mathematical Challenges and Ecology
Authors: Grace James B.
Published: July 2002

The article “Mathematical and computational challenges in population biology and ecosystems science” by Simon A. Levin
et al
. (
17 Jan., p. 334
) discusses exciting developments and challenges for studies of complex ecological systems, with special emphasis on simulation and analytical approaches. The authors highlight how increased computation capability is affecting our ability to tackle tough questions about complex system behavior. Another related area with similar challenges and advances, however, is the statistical analysis of data from complex systems. Extracting a clear understanding of how complex ecological systems operate will depend not only on our ability to simulate component processes but will also require us to go beyond traditional experimental approaches, which are limited in their scope, duration, and realism, for practical reasons.

Relative to their rapid adoption of simulation and analytic approaches, ecologists have been slow to exploit the most recent advances in multivariate analyses. Traditional statistical approaches have often distinguished between ANOVA/MANOVA (analysis of variance/multivariate analyses of variance) approaches—which have a limited capability to deal with multivariate, interacting factors—and descriptive multivariate methods such as principal component analysis, factor analysis, and multiple regression. With the development of new programs that use the ready availability of increased computing power, data analysts are examining more of the whole covariance structure that occurs in complex systems. Expanded capacity for covariance analysis within ANOVA as well as capabilities in structural equation modeling are leading toward more general and less restricted methods of analysis. In the forefront of this enterprise should be the difficult task of interpreting the results from these analyses. No doubt, mistakes will be made and major refinements required. Nonetheless, in order to make useful predictions about the behavior of ecological systems, the challenge of conducting and interpreting multivariate analyses of interacting components must be tackled with vigor.
Prevention of Vascular and Neural Dysfunction in Diabetic Rats by C-Peptide
Authors: Ido Y. , Vindigni A. , Chang K. , Stramm L. , Chance R. , Heath W. F. , DiMarchi R. D. , Di Cera E. , Williamson J. R.
Published: July 2002

C-peptide, a cleavage product from the processing of proinsulin to insulin, has been considered to possess little if any biological activity other than its participation in insulin synthesis. Injection of human C-peptide prevented or attenuated vascular and neural (electrophysiological) dysfunction and impaired Na
+
- and K
+
-dependent adenosine triphosphate activity in tissues of diabetic rats. Nonpolar amino acids in the midportion of the peptide were required for these biological effects. Synthetic reverse sequence (retro) and all–
d
–amino acid (enantio) C-peptides were equipotent to native C-peptide, which indicates that the effects of C-peptide on diabetic vascular and neural dysfunction were mediated by nonchiral interactions instead of stereospecific receptors or binding sites.
Evidence That Gene Amplification Underlies Adaptive Mutability of the Bacterial
lac
Operon
Authors: Andersson Dan I. , Slechta E. Susan , Roth John R.
Published: July 2002

Adaptive mutability is the apparent alteration in specificity or rate of mutability seen in bacteria during stress. A model is proposed by which gene amplification during selective growth can give the appearance of adaptive mutability without requiring any change in mutability. The model is based on two assumptions, that a mutant
lac
locus with residual function allows growth if its copy number is increased, and that true reversion events are made more likely by replication of chromosomes with many copies of the locus. Apparent directed mutability, its recombination requirement, and its apparent independence of cell growth are all accounted for by the model. Evidence is provided for the required residual function and gene amplification.
The Dusty Atmosphere of the Brown Dwarf Gliese 229B
Authors: Griffith Caitlin A. , Yelle Roger V. , Marley Mark S.
Published: July 2002

The brown dwarf Gliese 229B has an observable atmosphere too warm to contain ice clouds like those on Jupiter and too cool to contain silicate clouds like those on low-mass stars. These unique conditions permit visibility to higher pressures than possible in cool stars or planets. Gliese 229B's 0.85- to 1.0-micrometer spectrum indicates particulates deep in the atmosphere (10 to 50 bars) having optical properties of neither ice nor silicates. Their reddish color suggests an organic composition characteristic of aerosols in planetary stratospheres. The particles' mass fraction (10
−7
) agrees with a photochemical origin caused by incident radiation from the primary star and suggests the occurrence of processes native to planetary stratospheres.
High Direct Estimate of the Mutation Rate in the Mitochondrial Genome of
Caenorhabditis elegans
Authors: Denver Dee R. , Morris Krystalynne , Lynch Michael , Vassilieva Larissa L. , Thomas W. Kelley
Published: July 2002

Mutations in the mitochondrial genome have been implicated in numerous human genetic disorders and offer important data for phylogenetic, forensic, and population genetic studies. Using a long-term series of
Caenorhabditis elegans
mutation accumulation lines, we performed a wide-scale screen for mutations in the mitochondrial genome that revealed a mutation rate that is two orders of magnitude higher than previous indirect estimates, a highly biased mutational spectrum, multiple mutations affecting coding function, as well as mutational hotspots at homopolymeric nucleotide stretches.
Fluoroquinolone Transport by Human Monocytes: Characterization and Comparison to Other Cells of Myeloid Lineage
Authors: Bounds Steven J. , Nakkula Robin , Walters John D.
Published: July 2002

Human monocytes transport and accumulate ciprofloxacin and other fluoroquinolones. Although little is known about the mechanisms of transport, we expected monocytes to be similar to other cells of myeloid lineage. In the present study, monocyte fluoroquinolone transport was characterized and compared to the corresponding transport pathways of human polymorphonuclear leukocytes (PMNs) and HL-60 cells. Ciprofloxacin transport by monocytes was saturable, temperature dependent, sodium independent, and relatively insensitive to pH. Quiescent monocytes transported ciprofloxacin with a

K
m

of 171 μg/ml and a
V
max
of 32.7 ng/min/10
6
cells. Adenine competitively inhibited ciprofloxacin transport by quiescent monocytes (

K
i

= 3.8 mM), but nucleosides had no significant inhibitory effect. In all of these respects, transport by monocytes was similar to that observed for quiescent PMNs and immature HL-60 cells. Unlike PMNs, however, monocytes and immature HL-60 cells did not exhibit dramatically enhanced ciprofloxacin transport when activated by phorbol myristate acetate (PMA). Consistent with this finding, HL-60 cells committed to granulocytic differentiation exhibited a significant component of PMA-inducible ciprofloxacin transport activity, while HL-60 cells committed to monocytic differentiation did not. In PMNs, the PMA-inducible component of transport appeared to be mobilized from a granule compartment, since its activity could be modulated by agents that enhance or inhibit stimulated degranulation. Thus, quiescent monocytes, PMNs, and HL-60 cells take up ciprofloxacin via similar energy-dependent transport mechanisms. Unlike granulocytes, monocytes do not express a second, higher-affinity pathway for ciprofloxacin accumulation when they are activated by PMA.
Toward Antiviral Strategies That Resist Viral Escape
Authors: Endy Drew , Yin John
Published: July 2002

We studied the effect on viral growth of drugs targeting different virus functions using a computer simulation for the intracellular growth of bacteriophage T7. We found that drugs targeting components of negative-feedback loops gain effectiveness against mutant viruses that attenuate the drug-target interaction. The greater inhibition of such mutants than of the wild type suggests a drug design strategy that would hinder the development of drug resistance.
Mechanisms and Frequency of Resistance to Gatifloxacin in Comparison to AM-1121 and Ciprofloxacin in
Staphylococcus aureus
Authors: Ince Dilek , Hooper David C.
Published: July 2002

Gatifloxacin, an 8-methoxyfluoroquinolone, was found to be two- to fourfold more active against wild-type
Staphylococcus aureus
ISP794 than its desmethoxy derivative, AM-1121, and ciprofloxacin, another desmethoxy fluoroquinolone. Single
grlBA
mutations caused two- to fourfold increases in the MIC of gatifloxacin, and a single gyrase mutation was silent. Double mutations in
gyrA
and
grlA
or
grlB
caused a 32-fold increase in the MIC of gatifloxacin, in contrast to a 128-fold increase for ciprofloxacin and AM-1121. Overexpression of the NorA efflux pump had minimal effect on the MIC of gatifloxacin. The bactericidal activity of the three quinolones at four times the MIC differed only for a double mutant, with gatifloxacin exhibiting a killing pattern similar to that for ISP794, whereas ciprofloxacin and AM-1121 failed to show any killing. With gatifloxacin, selection of resistant mutants at twice the MIC was 100- to 1,000-fold less frequent than with the comparison quinolones, and mutants could rarely be selected at four times the MIC. The limit resistance in ISP74 was 512 times the MIC of gatifloxacin and 1,024 times the MICs of ciprofloxacin and AM-1121. Novel mutations in topoisomerase IV were selected in five of the six single-step mutants, three of which were shown to cause quinolone resistance by genetic studies. In conclusion, topoisomerase IV is the primary target of gatifloxacin. In contrast to comparison quinolones, mutations in both topoisomerase IV and gyrase are required for resistance to gatifloxacin by clinical breakpoints and do not abolish bactericidal effect, further supporting the benefit of the 8-methoxy substituent in gatifloxacin.
Differential Antifungal Activity of Isomeric Forms of Nystatin
Authors: Ostrosky-Zeichner Luis , Bazemore Scott , Paetznick Victor L. , Rodriguez Jose R. , Chen Enuo , Wallace Tom , Cossum Paul , Rex John H.
Published: July 2002

When nystatin is placed in RPMI and other biological fluids, there is loss of pure nystatin, with the development of two distinguishable chromatographic peaks, 1 and 2. Peak 1 appears identical to commercially prepared nystatin. By nuclear magnetic resonance (NMR) and mass spectral analysis, peak 2 appears to be an isomer of peak 1. The isomers are quantitatively and fully interconvertible. Formation of peak 2 is accelerated at a pH of >7.0 and ultimately reaches a near 55:45 (peak 1/peak 2 ratio) mixture. We sought to determine the relative activities of peaks 1 and 2 against
Candida
spp. Peak 2 consistently showed higher MICs when it was the predominant form during the experiment. Time-kill analyses showed that peak 2 required ≥8× the concentration of peak 1 to produce a modest and delayed killing effect, which was never of the same magnitude as that produced by peak 1. In both types of assays, the activity of peak 2 corresponded with intra-assay formation of peak 1. Both MIC measurements and time-kill analysis suggest that peak 2 has considerably less activity, if any at all, against
Candida
spp. Peak 2 may serve as a reservoir for peak 1.
Multiple-Dose Pharmacokinetics and Tolerability of Gemifloxacin Administered Orally to Healthy Volunteers
Authors: Allen Ann , Bygate Elizabeth , Vousden Marika , Oliver Stuart , Johnson Martin , Ward Christopher , Cheon Ae-Jin , Choo Youn Sung , Kim In-Chull
Published: July 2002

Gemifloxacin mesylate (SB-265805-S, LB-20304a) is a potent, novel fluoroquinolone agent with a broad spectrum of antibacterial activity. The pharmacokinetics and tolerability of oral gemifloxacin were characterized in two parallel group studies in healthy male volunteers after doses of 160, 320, 480, and 640 mg once daily for 7 days. Multiple serum or plasma and urine samples were collected on days 1 and 7 and were analyzed for gemifloxacin by high-performance liquid chromatography (HPLC)–fluorescence (study 1) or HPLC-mass spectrometry (study 2). Safety assessments included vital signs, 12-lead electrocardiogram (ECG) readings, hematology, clinical chemistry, urinalysis, and adverse experience monitoring. Gemifloxacin was rapidly absorbed, with a time to maximum concentration of approximately 1 h after dosing followed by a biexponential decline in concentration. Generally, maximum concentration and area under the concentration-time curve (AUC) increased linearly with dose after either single or repeat doses. Mean ± standard deviation values of AUC
0–τ
on day 7 were 4.92 ± 1.08, 9.06 ± 2.20, 12.2 ± 3.69, and 20.1 ± 3.67 μg·h/ml following 160-, 320-, 480-, and 640-mg doses, respectively. The terminal-phase half-life was approximately 7 to 8 h, independent of dose, and was similar following single and repeated administrations. There was minimal accumulation of gemifloxacin after multiple dosing. Approximately 20 to 30% of the administered dose was excreted unchanged in the urine. The renal clearance was 160 ml/min on average after single and multiple doses, which was slightly greater than the accepted glomerular filtration rate (approximately 120 ml/min). These data show that the pharmacokinetics of gemifloxacin are linear and independent of dose. Gemifloxacin was generally well tolerated, although one subject was withdrawn from the study after 6 days at 640 mg for mild, transient elevations of alanine aminotransferase and aspartate aminotransferase not associated with any clinical signs or symptoms. There were no other significant changes in clinical chemistry, hematology or urinalysis parameters, vital signs, or ECG readings. In conclusion, the results of these studies, combined with the antibacterial spectrum and potency, support the further investigation of once-daily administration of gemifloxacin for indications such as respiratory tract and urinary tract infections.
Structural Comparison of Three Types of Staphylococcal Cassette Chromosome
mec
Integrated in the Chromosome in Methicillin-Resistant
Staphylococcus aureus
Authors: Ito Teruyo , Katayama Yuki , Asada Kazumi , Mori Namiko , Tsutsumimoto Kanae , Tiensasitorn Chuntima , Hiramatsu Keiichi
Published: July 2002

The β-lactam resistance gene
mecA
of
Staphylococcus aureus
is carried by a novel mobile genetic element, designated staphylococcal cassette chromosome
mec
(SCC
mec
), identified in the chromosome of a Japanese methicillin-resistant
S. aureus
(MRSA) strain. We now report identification of two additional types of
mecA
-carrying genetic elements found in the MRSA strains isolated in other countries of the world. There were substantial differences in the size and nucleotide sequences between the elements and the SCC
mec
. However, new elements shared the chromosomal integration site with the SCC
mec
. Structural analysis of the new elements revealed that they possessed all of the salient features of the SCC
mec
: conserved terminal inverted repeats and direct repeats at the integration junction points, conserved genetic organization around the
mecA
gene, and the presence of cassette chromosome recombinase (
ccr
) genes responsible for the movements of SCC
mec
. The elements, therefore, were considered to comprise the SCC
mec
family of staphylococcal mobile genetic elements together with the previously identified SCC
mec
. Among 38 epidemic MRSA strains isolated in 20 countries, 34 were shown to possess one of the three typical SCC
mec
elements on the chromosome. Our findings indicated that there are at least three distinct MRSA clones in the world with different types of SCC
mec
in their chromosome.
Phenotypic Susceptibilities to Tenofovir in a Large Panel of Clinically Derived Human Immunodeficiency Virus Type 1 Isolates
Authors: Harrigan P. R. , Miller M. D. , McKenna P. , Brumme Z. L. , Larder B. A.
Published: July 2002

Tenofovir is a nucleotide analogue human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitor, and its oral prodrug, tenofovir disoproxil fumarate, has recently been approved for the treatment of HIV-1 infection in the United States. The objective of this study was to characterize the in vitro susceptibility profiles of a large panel of clinically derived HIV-1 isolates for tenofovir. The distribution of tenofovir susceptibilities in over 1,000 antiretroviral-naive, HIV-1-infected individuals worldwide was determined using the Virco Antivirogram assay. In addition, phenotypic susceptibilities to tenofovir and other RT inhibitors were determined in a panel of nearly 5,000 recombinant HIV-1 clinical isolates from predominantly treatment-experienced patients analyzed as a part of routine drug resistance testing. Greater than 97.5% of isolates from treatment-naive patients had tenofovir susceptibilities <3-fold above those of the wild-type controls by the Antivirogram. The clinically derived panel of 5,000 samples exhibited a broad range of antiretroviral drug susceptibilities, including 69, 43, and 16% having >10-fold-decreased susceptibilities to at least one, two, and three antiretroviral drug classes, respectively. Greater than 88% of these 5,000 clinical isolates were within the threefold susceptibility range for tenofovir, and >99% exhibited <10-fold-reduced susceptibilities to tenofovir. Decreased susceptibility to tenofovir was not directly associated with resistance to other RT inhibitors;r2values of log-log linear regression plots of susceptibility to tenofovir versus susceptibility to other RT inhibitors were <0.4. The results suggest that the majority of treatment-naive and treatment-experienced individuals harbor HIV that remains within the normal range of tenofovir susceptibilities and may be susceptible to tenofovir disoproxil fumarate therapy.
Method for Measuring Postantifungal Effect inAspergillusSpecies
Authors: Vitale Roxana G. , Mouton Johan W. , Afeltra Javier , Meis Jacques F. G. M. , Verweij Paul E.
Published: July 2002

An in vitro method for determination of postantifungal effect (PAFE) in molds was developed by using three isolates each ofAspergillus fumigatus,A. flavus,A. terreus,A. nidulans, andA. ustus. MICs of amphotericin B and itraconazole were determined by using National Committee for Clinical Laboratory Standards guidelines (M38-P). The inoculum was prepared in RPMI 1640 broth buffered with MOPS (morpholinepropanesulfonic acid) at pH 7.0, and conidia were exposed to amphotericin B and itraconazole at concentrations of 4, 1, and 0.25 times the MIC, each for 4, 2, and 1 h at 37°C. The same procedure was followed for controls with drug-free medium. Following exposure, the conidia were washed three times in saline and the numbers of CFU per milliliter were determined. Exposed and control conidia were then inoculated into microtitration plates and incubated at 37°C for 48 h in a spectrophotometer reader. The optical density (OD) was measured automatically at 10-min intervals, resulting in growth curves. PAFE was quantified by comparing three arbitrary points in the control growth curve, the first increase of OD and the points when 20 and 50% of the maximal growth were reached, with the growth curve of drug-exposed conidia. Amphotericin B induced PAFE inA. fumigatusat four times the MIC after 2 and 4 h of exposure ranging from 1.83 to 6.00 h and 9.33 to 10.80 h, respectively. Significantly shorter PAFEs or lack of PAFE was observed forA. terreus,A. ustus, andA. nidulans. Itraconazole did not induce measurable PAFE in theAspergillusisolates at any concentration or exposure time tested. Further studies are warranted to investigate the implications of PAFE in relation to clinical efficacy and dosing frequency.
Effects of Synthetic Siderophores on Proliferation ofPlasmodium falciparumin Infected Human Erythrocytes
Authors: Rotheneder Andrea , Fritsche Gernot , Heinisch Lothar , Möllmann Ute , Heggemann Susanne , Larcher Clara , Weiss Günter
Published: July 2002

Because iron is essential forPlasmodium falciparum, we investigated the in vitro potential of various synthetic siderophores to killP. falciparumin infected human erythrocytes. The substances with the most promising profile, i.e., low 50% lethal doses for plasmodia and minimum toxicity towards mammalian cells, were siderophores with an acylated monocatecholate or a triscatecholate as substituent.
Development of Real-Time PCR Assays for Rapid Detection of
Pfiesteria piscicida
and Related Dinoflagellates
Authors: Bowers Holly A. , Tengs Torstein , Glasgow Howard B. , Burkholder JoAnn M. , Rublee Parke A. , Oldach David W.
Published: July 2002

Pfiesteria
complex species are heterotrophic and mixotrophic dinoflagellates that have been recognized as harmful algal bloom species associated with adverse fish and human health effects along the East Coast of North America, particularly in its largest (Chesapeake Bay in Maryland) and second largest (Albermarle-Pamlico Sound in North Carolina) estuaries. In response to impacts on human health and the economy, monitoring programs to detect the organism have been implemented in affected areas. However, until recently, specific identification of the two toxic species known thus far,
Pfiesteria piscicida
and
P. shumwayae
(sp. nov.), required scanning electron microscopy (SEM). SEM is a labor-intensive process in which a small number of cells can be analyzed, posing limitations when the method is applied to environmental estuarine water samples. To overcome these problems, we developed a real-time PCR-based assay that permits rapid and specific identification of these organisms in culture and heterogeneous environmental water samples. Various factors likely to be encountered when assessing environmental samples were addressed, and assay specificity was validated through screening of a comprehensive panel of cultures, including the two recognized
Pfiesteria
species, morphologically similar species, and a wide range of other estuarine dinoflagellates. Assay sensitivity and sample stability were established for both unpreserved and fixative (acidic Lugol's solution)-preserved samples. The effects of background DNA on organism detection and enumeration were also explored, and based on these results, we conclude that the assay may be utilized to derive quantitative data. This real-time PCR-based method will be useful for many other applications, including adaptation for field-based technology.
Key Aromatic-Ring-Cleaving Enzyme, Protocatechuate 3,4-Dioxygenase, in the Ecologically Important Marine
Roseobacter
Lineage
Authors: Buchan Alison , Collier Lauren S. , Neidle Ellen L. , Moran Mary Ann
Published: July 2002

Aromatic compound degradation in six bacteria representing an ecologically important marine taxon of the α-proteobacteria was investigated. Initial screens suggested that isolates in the
Roseobacter
lineage can degrade aromatic compounds via the β-ketoadipate pathway, a catabolic route that has been well characterized in soil microbes. Six
Roseobacter
isolates were screened for the presence of protocatechuate 3,4-dioxygenase, a key enzyme in the β-ketoadipate pathway. All six isolates were capable of growth on at least three of the eight aromatic monomers presented (anthranilate, benzoate,
p
-hydroxybenzoate, salicylate, vanillate, ferulate, protocatechuate, and coumarate). Four of the
Roseobacter
group isolates had inducible protocatechuate 3,4-dioxygenase activity in cell extracts when grown on
p
-hydroxybenzoate. The
pcaGH
genes encoding this ring cleavage enzyme were cloned and sequenced from two isolates,
Sagittula stellata
E-37 and isolate Y3F, and in both cases the genes could be expressed in
Escherichia coli
to yield dioxygenase activity. Additional genes involved in the protocatechuate branch of the β-ketoadipate pathway (
pcaC
,
pcaQ
, and
pobA
) were found to cluster with
pcaGH
in these two isolates. Pairwise sequence analysis of the
pca
genes revealed greater similarity between the two
Roseobacter
group isolates than between genes from either
Roseobacter
strain and soil bacteria. A degenerate PCR primer set targeting a conserved region within PcaH successfully amplified a fragment of
pcaH
from two additional
Roseobacter
group isolates, and Southern hybridization indicated the presence of
pcaH
in the remaining two isolates. This evidence of protocatechuate 3,4-dioxygenase and the β-ketoadipate pathway was found in all six
Roseobacter
isolates, suggesting widespread abilities to degrade aromatic compounds in this marine lineage.
Exogenous Isolation of Antibiotic Resistance Plasmids from Piggery Manure Slurries Reveals a High Prevalence and Diversity of IncQ-Like Plasmids
Authors: Smalla Kornelia , Heuer Holger , Götz Antje , Niemeyer Dagmar , Krögerrecklenfort Ellen , Tietze Erhard
Published: July 2002

Antibiotic resistance plasmids were exogenously isolated in biparental matings with piggery manure bacteria as plasmid donors in
Escherichia coli
CV601 and
Pseudomonas putida
UWC1 recipients. Surprisingly, IncQ-like plasmids were detected by dot blot hybridization with an IncQ
oriV
probe in several
P. putida
UWC1 transconjugants. The capture of IncQ-like plasmids in biparental matings indicates not only their high prevalence in manure slurries but also the presence of efficiently mobilizing plasmids. In order to elucidate unusual hybridization data (weak or no hybridization with IncQ
repB
or IncQ
oriT
probes) four IncQ-like plasmids (pIE1107, pIE1115, pIE1120, and pIE1130), each representing a different
Eco
RV restriction pattern, were selected for a more thorough plasmid characterization after transfer into
E. coli
K-12 strain DH5α by transformation. The characterization of the IncQ-like plasmids revealed an astonishingly high diversity with regard to phenotypic and genotypic properties. Four different multiple antibiotic resistance patterns were found to be conferred by the IncQ-like plasmids. The plasmids could be mobilized by the RP4 derivative pTH10 into
Acinetobacter
sp.,
Ralstonia eutropha
,
Agrobacterium tumefaciens
, and
P. putida
, but they showed diverse patterns of stability under nonselective growth conditions in different host backgrounds. Incompatibility testing and PCR analysis clearly revealed at least two different types of IncQ-like plasmids. PCR amplification of total DNA extracted directly from different manure samples and other environments indicated the prevalence of both types of IncQ plasmids in manure, sewage, and farm soil. These findings suggest that IncQ plasmids play an important role in disseminating antibiotic resistance genes.
Monitoring Bacterial Transport by Stable Isotope Enrichment of Cells
Authors: Holben William E. , Ostrom Peggy H.
Published: July 2002

Understanding the transport and behavior of bacteria in the environment has broad implications in diverse areas, ranging from agriculture to groundwater quality, risk assessment, and bioremediation. The ability to reliably track and enumerate specific bacterial populations in the context of native communities and environments is key to developing this understanding. We report a novel bacterial tracking approach, based on altering the stable carbon isotope value (δ
13
C) of bacterial cells, which provides specific and sensitive detection and quantification of those cells in environmental samples. This approach was applied to the study of bacterial transport in saturated porous media. The transport of introduced organisms was indicated by mass spectrometric analysis of groundwater samples, where the presence of
13
C-enriched bacteria resulted in increased δ
13
C values of the samples, allowing specific and sensitive detection and enumeration of the bacteria of interest. We demonstrate the ability to produce highly
13
C-enriched bacteria, present data indicating that results obtained with this approach accurately represent intact introduced bacteria, and include field data on the use of this stable isotope approach to monitor in situ bacterial transport. This detection strategy allows sensitive detection of an introduced, unmodified bacterial strain in the presence of the indigenous bacterial community, including itself in its unenriched form.
Dynamics of Bacterial Community Composition and Activity during a Mesocosm Diatom Bloom
Authors: Riemann Lasse , Steward Grieg F. , Azam Farooq
Published: July 2002

Bacterial community composition, enzymatic activities, and carbon dynamics were examined during diatom blooms in four 200-liter laboratory seawater mesocosms. The objective was to determine whether the dramatic shifts in growth rates and ectoenzyme activities, which are commonly observed during the course of phytoplankton blooms and their subsequent demise, could result from shifts in bacterial community composition. Nutrient enrichment of metazoan-free seawater resulted in diatom blooms dominated by aThalassiosirasp., which peaked 9 days after enrichment (≈24 μg of chlorophyllaliter−1). At this time bacterial abundance abruptly decreased from 2.8 × 106to 0.75 × 106ml−1, and an analysis of bacterial community composition, by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments, revealed the disappearance of three dominant phylotypes. Increased viral and flagellate abundances suggested that both lysis and grazing could have played a role in the observed phylotype-specific mortality. Subsequently, new phylotypes appeared and bacterial production, abundance, and enzyme activities shifted from being predominantly associated with the <1.0-μm size fraction towards the >1.0-μm size fraction, indicating a pronounced microbial colonization of particles. Sequencing of DGGE bands suggested that the observed rapid and extensive colonization of particulate matter was mainly by specialized α-Proteobacteria- andCytophagales-related phylotypes. These particle-associated bacteria had high growth rates as well as high cell-specific aminopeptidase, β-glucosidase, and lipase activities. Rate measurements as well as bacterial population dynamics were almost identical among the mesocosms indicating that the observed bacterial community dynamics were systematic and repeatable responses to the manipulated conditions.
Transformation of
Acinetobacter
sp. Strain BD413(pFG4Δ
nptII
) with Transgenic Plant DNA in Soil Microcosms and Effects of Kanamycin on Selection of Transformants
Authors: Nielsen Kaare M. , van Elsas Jan D. , Smalla Kornelia
Published: July 2002

Here we show that horizontal transfer of DNA, extracted from transgenic sugar beets, to bacteria, based on homologous recombination, can occur in soil. Restoration of a 317-bp-deleted
nptII
gene in
Acinetobacter
sp. strain BD413(pFG4) cells incubated in sterile soil microcosms was detected after addition of nutrients and transgenic plant DNA encoding a functional
nptII
gene conferring bacterial kanamycin resistance. Selective effects of the addition of kanamycin on the population dynamics of
Acinetobacter
sp. cells in soil were found, and high concentrations of kanamycin reduced the CFU of
Acinetobacter
sp. cells from 10
9
CFU/g of soil to below detection. In contrast to a chromosomal
nptII
-encoded kanamycin resistance, the pFG4-generated resistance was found to be unstable over a 31-day incubation period in vitro.
Bioconversion of Ferulic Acid into Vanillic Acid by Means of a Vanillate-Negative Mutant of
Pseudomonas fluorescens
Strain BF13
Authors: Civolani Claudio , Barghini Paolo , Roncetti Anna Rita , Ruzzi Maurizio , Schiesser Alma
Published: July 2002

From a ferulic-acid-degrading
Pseudomonas fluorescens
strain (BF13), we have isolated a transposon mutant, which retained the ability to bioconvert ferulic acid into vanillic acid but lost the ability to further degrade the latter acid. The mutant, BF13-97, was very stable, and therefore it was suitable to be used as a biocatalyst for the preparative synthesis of vanillic acid from ferulic acid. By use of resting cells we determined the effect on the bioconversion rate of several parameters, such as the addition of nutritional factors, the concentration of the biomass, and the carbon source on which the biomass was grown. The optimal yield of vanillic acid was obtained with cells pregrown on M9 medium containing
p
-coumaric acid (0.1% [wt/vol]) as a sole carbon source and yeast extract (0.001% [wt/vol]) as a source of nutritional factors. Under these conditions, 1 mg (wet weight) of biomass produced 0.23 mg of vanillic acid per h. The genomic region of BF13-97 flanking the transposon's site of insertion was cloned and sequenced revealing two open reading frames of 1,062 (
vanA
) and 954 (
vanB
) bp, respectively. The
van
genes are organized in a cluster and encode the subunits of the vanillate-
O
-demethylase, which catalyzes the first step of the vanillate catabolism. Amino acid sequences deduced from
vanA
and
vanB
genes were shown to have high identity with known VanAs and VanBs from
Pseudomonas
and
Acinetobacter
spp. Highly conserved regions known to exist in class IA oxygenases were also found in the vanillate-
O
-demethylase components from
P. fluorescens
BF13. The terminal oxygenase VanA is characterized by a conserved Rieske-type [2Fe-2S]
R
ligand center. The reductase VanB contains a plant-type ferredoxin [2Fe-2S]
Fd
, flavin mononucleotide, and NAD-ribose binding domains which are located in its C-terminal and N-terminal halves, respectively. Transfer of wild-type
vanAB
genes to BF13-97 complemented this mutant, which recovered its ability to grow on either vanillic or ferulic acid.
Sequence Analysis and Initial Characterization of Two Isozymes of Hydroxylaminobenzene Mutase from
Pseudomonas pseudoalcaligenes
JS45
Authors: Davis John K. , Paoli George C. , He Zhongqi , Nadeau Lloyd J. , Somerville Charles C. , Spain Jim C.
Published: July 2002

Pseudomonas pseudoalcaligenes
JS45 grows on nitrobenzene by a partially reductive pathway in which the intermediate hydroxylaminobenzene is enzymatically rearranged to 2-aminophenol by hydroxylaminobenzene mutase (HAB mutase). The properties of the enzyme, the reaction mechanism, and the evolutionary origin of the gene(s) encoding the enzyme are unknown. In this study, two open reading frames (
habA
and
habB
), each encoding an HAB mutase enzyme, were cloned from a
P. pseudoalcaligenes
JS45 genomic library and sequenced. The open reading frames encoding HabA and HabB are separated by 2.5 kb and are divergently transcribed. The deduced amino acid sequences of HabA and HabB are 44% identical. The HAB mutase specific activities in crude extracts of
Escherichia coli
clones synthesizing either HabA or HabB were similar to the specific activities of extracts of strain JS45 grown on nitrobenzene. HAB mutase activity in
E. coli
extracts containing HabB withstood heating at 85°C for 10 min, but extracts containing HabA were inactivated when they were heated at temperatures above 60°C. HAB mutase activity in extracts of
P. pseudoalcaligenes
JS45 grown on nitrobenzene exhibited intermediate temperature stability. Although both the
habA
gene and the
habB
gene conferred HAB mutase activity when they were separately cloned and expressed in
E. coli
, reverse transcriptase PCR analysis indicated that only
habA
is transcribed in
P. pseudoalcaligenes
JS45. A mutant strain derived from strain JS45 in which the
habA
gene was disrupted was unable to grow on nitrobenzene, which provided physiological evidence that HabA is involved in the degradation of nitrobenzene. A strain in which
habB
was disrupted grew on nitrobenzene. Gene Rv3078 of
Mycobacterium tuberculosis
H37Rv encodes a protein whose deduced amino acid sequence is 52% identical to the HabB amino acid sequence.
E. coli
containing
M. tuberculosis
gene Rv3078 cloned into pUC18 exhibited low levels of HAB mutase activity. Sequences that exhibit similarity to transposable element sequences are present between
habA
and
habB
, as well as downstream of
habB
, which suggests that horizontal gene transfer resulted in acquisition of one or both of the
hab
genes.
Duplication of a Truncated Paralog of the Family B DNA Polymerase Gene
Aa-polB
in the
Agrocybe aegerita
Mitochondrial Genome
Authors: Barroso Gerard , Bois Frederic , Labarère Jacques
Published: July 2002

The
Agrocybe aegerita
mitochondrial genome contains a truncated family B DNA polymerase gene (
Aa-polB P1
) whose nucleotide sequence is 86% identical to the previously described and potentially functional
Aa-polB
gene. A tRNA
Met
gene occurs at the 3′ end of the
Aa-polB P1
gene. The
Aa-polB P1
gene could result from reverse transcription of an
Aa-polB
mRNA primed by a tRNA
Met
followed by the integration of the cDNA after recombination at the mitochondrial tRNA locus. Two naturally occurring alleles of
Aa-polB P1
carry one or two copies of the disrupted sequence. In strains with two copies of
Aa-polB P1
, these copies are inverted relative to one another and separated by a short sequence carrying the tRNA
Met
gene. Both
A. aegerita
mitochondrial family B DNA polymerases were found to be related to other family B DNA polymerases (36 to 53% amino acid similarity), including the three enzymes of the archaebacterium
Sulfolobus solfataricus
. If mitochondria originated from a fusion between a
Clostridium
-like eubacterium and a
Sulfolobus
-like archaebacterium, then the
A. aegerita
family B DNA polymerase genes could be remnants of the archaebacterial genes.
Purification and Characterization of Two Different α-l-Rhamnosidases, RhaA and RhaB, fromAspergillus aculeatus
Authors: Manzanares Paloma , van den Broeck Hetty C. , de Graaff Leo H. , Visser Jaap
Published: July 2002

Two proteins exhibiting α-l-rhamnosidase activity, RhaA and RhaB, were identified upon fractionation and purification of a culture filtrate fromAspergillus aculeatusgrown on hesperidin. Both proteins were shown to be N glycosylated and had molecular masses of 92 and 85 kDa, of which approximately 24 and 15%, respectively, were contributed by carbohydrate. RhaA and RhaB, optimally active at pH 4.5 to 5, showedKmandVmaxvalues of 2.8 mM and 24 U/mg (RhaA) and 0.30 mM and 14 U/mg (RhaB) when tested forp-nitrophenyl-α-l-rhamnopyranoside. Both enzymes were able to hydrolyze α-1,2 and α-1,6 linkages to β-d-glucosides. Using polyclonal antibodies, the corresponding cDNA of both α-l-rhamnosidases,rhaAandrhaB, was cloned. On the basis of the amino acid sequences derived from the cDNA clones, both proteins are highly homologous (60% identity).
Two Distinct Hemolytic Activities in
Xenorhabdus nematophila
Are Active against Immunocompetent Insect Cells
Authors: Brillard Julien , Ribeiro Carlos , Boemare Noël , Brehélin Michel , Givaudan Alain
Published: July 2002

Xenorhabdus
spp. and
Photorhabdus
spp. are major insect bacterial pathogens symbiotically associated with nematodes. These bacteria are transported by their nematode hosts into the hemocoel of the insect prey, where they proliferate within hemolymph. In this work we report that wild strains belonging to different species of both genera are able to produce hemolysin activity on blood agar plates. Using a hemocyte monolayer bioassay, cytolytic activity against immunocompetent cells from the hemolymph of
Spodoptera littoralis
(Lepidoptera: Noctuidae) was found only in supernatants of
Xenorhabdus
; none was detected in supernatants of various strains of
Photorhabdus
. During in vitro bacterial growth of
Xenorhabdus nematophila
F1, two successive bursts of cytolytic activity were detected. The first extracellular cytolytic activity occurred when bacterial cells reached the stationary phase. It also displayed a hemolytic activity on sheep red blood cells, and it was heat labile. Among insect hemocyte types, granulocytes were the preferred target. Lysis of hemocytes by necrosis was preceded by a dramatic vacuolization of the cells. In contrast the second burst of cytolytic activity occurred late during stationary phase and caused hemolysis of rabbit red blood cells, and insect plasmatocytes were the preferred target. This second activity is heat resistant and produced shrinkage and necrosis of hemocytes. Insertional inactivation of
flhD
gene in
X. nematophila
leads to the loss of hemolysis activity on sheep red blood cells and an attenuated virulence phenotype in
S. littoralis
(A. Givaudan and A. Lanois, J. Bacteriol. 182:107–115, 2000). This mutant was unable to produce the early cytolytic activity, but it always displayed the late cytolytic effect, preferably active on plasmatocytes. Thus,
X. nematophila
produced two independent cytolytic activities against different insect cell targets known for their major role in cellular immunity.
Differentiation of Two Bovine Lentiviruses by a Monoclonal Antibody on the Basis of Epitope Specificity
Authors: Zheng Ling , Zhang Shucheng , Wood Charles , Kapil Sanjay , Wilcox Graham E. , Loughin Thomas A. , Minocha H. C.
Published: July 2002

Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are bovine lentiviruses that are closely related genetically. A recombinant fusion protein containing the capsid protein of BIV expressed in
Escherichia coli
was used to immunize mice and produce monoclonal antibodies. Six hybridomas specific for BIV capsid protein were identified, and one antibody, designated 10H1, was characterized further. Competitive binding assays were performed to analyze the topography of antigenic determinants by enzyme-linked immunosorbent assay and demonstrated the existence of at least three distinct antigenic determinants on capsid protein. The monoclonal antibody reacted specifically with both BIV capsid and the recombinant fusion protein in Western immunoblot analyses. However, it did not react with the recombinant capsid fusion protein of JDV, indicating that BIV contains at least one unique epitope in the capsid protein that is absent in JDV. Further mapping of the epitope by chemical cleavage analysis identified that the epitope is located at the 6.4-kDa N terminus of the 29-kDa capsid protein. This monoclonal antibody assay will be valuable for distinguishing the two closely related lentiviruses by Western blotting.