bluuebunny/crossref_metadata_2025 huggingface dataset:
Controlling Molecular Self-Organization: Formation of Nanometer-Scale Spheres and Tubules
Authors: Orr G. William , Barbour Leonard J. , Atwood Jerry L.
Published: July 2002

Amphiphilic polyhedron-shaped
p
-sulfonatocalix[4]arene building blocks, which have been previously shown to assemble into bilayers in an antiparallel fashion, have been assembled in a parallel alignment into spherical and helical tubular structures by the addition of pyridine
N
-oxide and lanthanide ions. Crystallographic studies revealed how metal ion coordination and substrate recognition direct the formation of these supramolecular assemblies. The addition of greater amounts of pyridine
N
-oxide changed the curvature of the assembling surface and resulted in the formation of extended tubules.
Requirement of Circadian Genes for Cocaine Sensitization in
Drosophila
Authors: Andretic Rozi , Chaney Sarah , Hirsh Jay
Published: July 2002

The circadian clock consists of a feedback loop in which clock genes are rhythmically expressed, giving rise to cycling levels of RNA and proteins. Four of the five circadian genes identified to date influence responsiveness to freebase cocaine in the fruit fly,
Drosophila melanogaster
. Sensitization to repeated cocaine exposures, a phenomenon also seen in humans and animal models and associated with enhanced drug craving, is eliminated in flies mutant for
period
,
clock
,
cycle
, and
doubletime
, but not in flies lacking the gene
timeless
. Flies that do not sensitize owing to lack of these genes do not show the induction of tyrosine decarboxylase normally seen after cocaine exposure. These findings indicate unexpected roles for these genes in regulating cocaine sensitization and indicate that they function as regulators of tyrosine decarboxylase.
Differential Stimulation of PKC Phosphorylation of Potassium Channels by ZIP1 and ZIP2
Authors: Gong Jianping , Xu Jia , Bezanilla Magdalena , Huizen Rika van , Derin Rachel , Li Min
Published: July 2002

Targeting of protein modification enzymes is a key biochemical step to achieve specific and effective posttranslational modifications. Two alternatively spliced ZIP1 and ZIP2 proteins are described, which bind to both Kvβ2 subunits of potassium channel and protein kinase C (PKC) ζ, thereby acting as a physical link in the assembly of PKCζ-ZIP-potassium channel complexes. ZIP1 and ZIP2 differentially stimulate phosphorylation of Kvβ2 by PKCζ. They also interact to form heteromultimers, which allows for a hybrid stimulatory activity to PKCζ. Finally, ZIP1 and ZIP2 coexist in the same cell type and are elevated differentially by neurotrophic factors. These results provide a mechanism for specificity and regulation of PKCζ-targeted phosphorylation.
Collisionally Processed Rocks on Mars
Authors: Hörz Friedrich , Cintala Mark J. , Rochelle W. C. , Kirk B.
Published: July 2002

The Pathfinder landing site on Mars has boulders that may be cratered (Stimpy), split (Chimp), fragmented (Book End and Flat Top), or otherwise partly destroyed (Yogi and Frog) by collisional processes. Atmospheric-entry calculations show that centimeter-sized projectiles survive passage through the martian atmosphere and encounter the surface of Mars at velocities of a few kilometers per second. Craters less than 1 meter in diameter may contribute to the evolution of the martian surface and its soils.
Laminar Ceramics That Exhibit a Threshold Strength
Authors: Rao M. P. , Sánchez-Herencia A. J. , Beltz G. E. , McMeeking R. M. , Lange F. F.
Published: July 2002

Thin compressive layers within a laminar ceramic arrest large cracks (surface and internal) and produce a threshold strength. This phenomenon increases the damage tolerance of ceramics and will allow engineers to design reliable ceramic components for structural applications. The stress intensity factor derived for a crack sandwiched between two compressive layers suggests that the threshold strength is proportional to the residual compressive stress and the thickness of the compressive layer and is inversely proportional to the distance between the compressive layers. Laminates composed of thick alumina layers (605 ± 11 micrometers) and thin mullite/alumina compressive layers (37 ± 1.4 micrometers) fabricated for this study had a threshold strength of 482 ± 20 megapascals, in fair agreement with the theory.
Why Do Kids Say Goed and Brang?
Authors: McClelland James L. , Seidenberg Mark S.
Published: July 2002

Words and Rules
The Ingredients of Language. Steven Pinker. Basic Books, New York, 1999. 362 pp. $26, C$39.50. ISBN 0-465-07269-0.

Pinker presents a popular exposition of his research into how children learn language. Although his account, focused on English past-tense verb forms, is engaging and informative, our reviewers argue that it does not grant neural network models the importance they warrant.
The Shape of 433 Eros from the NEAR-Shoemaker Laser Rangefinder
Authors: Zuber Maria T. , Smith David E. , Cheng Andrew F. , Garvin James B. , Aharonson Oded , Cole Timothy D. , Dunn Peter J. , Guo Yanping , Lemoine Frank G. , Neumann Gregory A. , Rowlands David D. , Torrence Mark H.
Published: July 2002

Measurements from the Near Earth Asteroid Rendezvous (NEAR)–Shoemaker Laser Rangefinder (NLR) indicate that asteroid 433 Eros is a consolidated body with a complex shape dominated by collisions. The offset between the asteroid's center of mass and center of figure indicates a small deviation from a homogeneous internal structure that is most simply explained by variations in mechanical structure. Regional-scale relief and slope distributions show evidence for control of topography by a competent substrate. Impact crater morphology is influenced by both gravity and structural control. Small-scale topography reveals ridges and grooves that may be generated by impact-related fracturing.
Widespread Origins of Domestic Horse Lineages
Authors: Vilà Carles , Leonard Jennifer A. , Götherström Anders , Marklund Stefan , Sandberg Kaj , Lidén Kerstin , Wayne Robert K. , Ellegren Hans
Published: July 2002

Domestication entails control of wild species and is generally regarded as a complex process confined to a restricted area and culture. Previous DNA sequence analyses of several domestic species have suggested only a limited number of origination events. We analyzed mitochondrial DNA (mtDNA) control region sequences of 191 domestic horses and found a high diversity of matrilines. Sequence analysis of equids from archaeological sites and late Pleistocene deposits showed that this diversity was not due to an accelerated mutation rate or an ancient domestication event. Consequently, high mtDNA sequence diversity of horses implies an unprecedented and widespread integration of matrilines and an extensive utilization and taming of wild horses. However, genetic variation at nuclear markers is partitioned among horse breeds and may reflect sex-biased dispersal and breeding.
Stereotyped Position of Local Synaptic Targets in Neocortex
Authors: Kozloski James , Hamzei-Sichani Farid , Yuste Rafael
Published: July 2002

The microcircuitry of the mammalian neocortex remains largely unknown. Although the neocortex could be composed of scores of precise circuits, an alternative possibility is that local connectivity is probabilistic or even random. To examine the precision and degree of determinism in the neocortical microcircuitry, we used optical probing to reconstruct microcircuits in layer 5 from mouse primary visual cortex. We stimulated “trigger” cells, isolated from a homogenous population of corticotectal pyramidal neurons, while optically detecting “follower” neurons directly driven by the triggers. Followers belonged to a few selective anatomical classes with stereotyped physiological and synaptic responses. Moreover, even the position of the followers appeared determined across animals. Our data reveal precisely organized cortical microcircuits.
Plasmid-Mediated Florfenicol Resistance Encoded by the
floR
Gene in
Escherichia coli
Isolated from Cattle
Authors: Cloeckaert Axel , Baucheron Sylvie , Flaujac Geraldine , Schwarz Stefan , Kehrenberg Corinna , Martel Jean-Louis , Chaslus-Dancla Elisabeth
Published: July 2002

A florfenicol resistance gene almost identical to
floR
of
Salmonella enterica
serovar Typhimurium DT104 was detected on 110- to 125-kb plasmids in
Escherichia coli
isolates of animal origin. Analysis of the
floR
gene flanking regions of one of the plasmids showed that they were different from those encountered in
S. enterica
serovar Typhimurium DT104.
Two New Mechanisms of Macrolide Resistance in Clinical Strains of
Streptococcus pneumoniae
from Eastern Europe and North America
Authors: Tait-Kamradt A. , Davies T. , Appelbaum P. C. , Depardieu F. , Courvalin P. , Petitpas J. , Wondrack L. , Walker A. , Jacobs M. R. , Sutcliffe J.
Published: July 2002

Resistance to macrolides in pneumococci is generally mediated by methylation of 23S rRNA via
erm
(B) methylase which can confer a macrolide (M)-, lincosamide (L)-, and streptogramin B (S
B
)-resistant (MLS
B
) phenotype or by drug efflux via
mef
(A) which confers resistance to 14- and 15-membered macrolides only. We studied 20 strains with unusual ML or MS
B
phenotypes which did not harbor
erm
(B) or
mef
(A). The strains had been isolated from patients in Eastern Europe and North America from 1992 to 1998. These isolates were found to contain mutations in genes for either 23S rRNA or ribosomal proteins. Three strains from the United States with an ML phenotype, each representing a different clone, were characterized as having an A2059G (
Escherichia coli
numbering) change in three of the four 23S rRNA alleles. Susceptibility to macrolides and lincosamides decreased as the number of alleles in isogenic strains containing A2059G increased. Sixteen MS
B
strains from Eastern Europe were found to contain a 3-amino-acid substitution (
69
GTG
71
to TPS) in a highly conserved region of the ribosomal protein L4 (
63
KPWRQK
GTG
RAR
74
). These strains formed several distinct clonal types. The single MS
B
strain from Canada contained a 6-amino-acid L4 insertion (
69
GTG
REKGTG
RAR), which impacted growth rate and also conferred a 500-fold increase in MIC on the ketolide telithromycin. These macrolide resistance mechanisms from clinical isolates are similar to those recently described for laboratory-derived mutants.
Effect of Proinflammatory Cytokines on the Interplay between Roxithromycin, HMR 3647, or HMR 3004 and Human Polymorphonuclear Neutrophils
Authors: Vazifeh D. , Bryskier A. , Labro M. T.
Published: July 2002

Cytokines, the hallmarks of infectious and inflammatory diseases, modify phagocyte activities and thus may interfere with the immunomodulating properties of antibacterial agents. We have investigated whether various proinflammatory cytokines (interleukin 1 [IL-1], IL-6, IL-8, gamma interferon, tumor necrosis factor alpha [TNF-α], and granulocyte-macrophage colony-stimulating factor [GM-CSF]) modify two macrolide properties, i.e., inhibition of oxidant production by polymorphonuclear neutrophils (PMN) and cellular uptake. Roxithromycin and two ketolides, HMR 3647 and HMR 3004, were chosen as the test agents. TNF-α and GM-CSF (but not the other cytokines) decreased the inhibitory effect of HMR 3647 only on oxidant production by PMN. Fifty percent inhibitory concentrations were, however, in the same range in control and cytokine-treated cells (about 60 to 70 μg/ml), suggesting that HMR 3647 acts downstream of the priming effect of cytokines. In contrast, the impairment of oxidant production by roxithromycin and HMR 3004 was unchanged (or increased) in cytokine-treated cells. This result suggests that HMR 3004 (the strongest inhibitory drug, likely owing to its quinoline side chain) and roxithromycin act on a cellular target upstream of cytokine action. In addition, TNF-α and GM-CSF significantly (albeit moderately) impaired (by about 20%) the uptake of the three molecules by PMN. The inhibitory effect of these two cytokines seems to be related to activation of the p38 mitogen-activated protein kinase. Our data also illuminate the mechanism underlying macrolide uptake: protein kinase A- and tyrosine kinase-dependent phosphorylation seems to be necessary for optimal uptake, while protein kinase C activation impairs it. The relevance of our data to the clinical setting requires further investigations, owing to the complexity of the cytokine cascade during infection and inflammation.
Antifungal Activity of Amphotericin B Cochleates against
Candida albicans
Infection in a Mouse Model
Authors: Zarif Leila , Graybill John R. , Perlin David , Najvar Laura , Bocanegra Rosie , Mannino Raphael J.
Published: July 2002

Cochleates are lipid-based supramolecular assemblies composed of natural products, negatively charged phospholipid, and a divalent cation. Cochleates can encapsulate amphotericin B (AmB), an important antifungal drug. AmB cochleates (CAMB) have a unique shape and the ability to target AmB to fungi. The minimal inhibitory concentration and the minimum lethal concentration against
Candida albicans
are similar to that for desoxycholate AmB (DAMB; Fungizone). In vitro, CAMB induced no hemolysis of human red blood cells at concentrations of as high as 500 μg of AmB/ml, and DAMB was highly hemolytic at 10 μg of AmB/ml. CAMB protect ICR mice infected with
C. albicans
when the agent is administered intraperitoneally at doses of as low as 0.1 mg/kg/day. In a tissue burden study, CAMB, DAMB, and AmBisome (liposomal AmB; LAMB) were effective in the kidneys, but in the spleen CAMB was more potent than DAMB at 1 mg/kg/day and was equivalent to LAMB at 10 mg/kg/day. In summary, CAMB are highly effective in treating murine candidiasis and compare well with AmBisome and AmB.
Antiviral
l
-Nucleosides Specific for Hepatitis B Virus Infection
Authors: Bryant Martin L. , Bridges Edward G. , Placidi Laurent , Faraj Abdesslem , Loi Anna-Giulia , Pierra Claire , Dukhan David , Gosselin Gilles , Imbach Jean-Louis , Hernandez Brenda , Juodawlkis Amy , Tennant Bud , Korba Brent , Cote Paul , Marion Pat , Cretton-Scott Erika , Schinazi Raymond F. , Sommadossi Jean-Pierre
Published: July 2002

A unique series of simple “unnatural” nucleosides has been discovered to inhibit hepatitis B virus (HBV) replication. Through structure-activity analysis it was found that the 3′-OH group of the β-
l
-2′-deoxyribose of the β-
l-
2′-deoxynucleoside confers specific antihepadnavirus activity. The unsubstituted nucleosides β-
l-
2′-deoxycytidine, β-
l
-thymidine, and β-
l
-2′-deoxyadenosine had the most potent, selective, and specific antiviral activity against HBV replication. Human DNA polymerases (α, β, and γ) and mitochondrial function were not affected. In the woodchuck model of chronic HBV infection, viral load was reduced by as much as 10
8
genome equivalents/ml of serum and there was no drug-related toxicity. In addition, the decline in woodchuck hepatitis virus surface antigen paralleled the decrease in viral load. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.
Identification and Expression of Cephamycinase
bla
CMY
Genes in
Escherichia coli
and
Salmonella
Isolates from Food Animals and Ground Meat
Authors: Zhao Shaohua , White David G. , McDermott Patrick F. , Friedman Sharon , English Linda , Ayers Sherry , Meng Jianghong , Maurer John J , Holland Robert , Walker Robert D.
Published: July 2002

Twenty-one
Salmonella
and 54
Escherichia coli
isolates, recovered from food animals and retail ground meats, that exhibited decreased susceptibilities to ceftiofur and ceftriaxone were shown to possess a
bla
CMY
gene. The
bla

CMY
-
4

gene was identified in an
E. coli
isolate recovered from retail chicken and was further shown to be responsible for resistance to cephalothin, ampicillin, and amoxicillin-clavulanic acid and elevated MICs of ceftriaxone, cefoxitin, and ceftiofur.
Antimicrobial Activity of Quinupristin-Dalfopristin Combined with Other Antibiotics against Vancomycin-Resistant Enterococci
Authors: Eliopoulos G. M. , Wennersten C. B.
Published: July 2002

Interactions between quinupristin-dalfopristin and six other antimicrobials were examined by checkerboard arrays against 50 clinical isolates of vancomycin-resistant
Enterococcus faecium
selected to represent a range of susceptibilities to individual agents. Unequivocal synergistic or antagonistic interactions at clinically relevant concentrations were infrequently encountered when the streptogramin was combined with chloramphenicol, ampicillin, imipenem, vancomycin, or teicoplanin. Combinations with doxycycline resulted in synergistic inhibition in 36% of checkerboards. Against 10 strains of
Enterococcus faecalis
, synergistic interactions were found when quinupristin-dalfopristin was combined with doxycycline (four strains), either glycopeptide (three strains), or ampicillin (two strains). Combination with quinupristin-dalfopristin increased the ampicillin MIC from 1 to 4 μg/ml for one strain. For 10 strains of
E. faecium
, interactions were also assessed by time-kill methods using concentrations of the agents attainable in human serum. Most of these antimicrobials augmented killing by quinupristin-dalfopristin to a minor degree. Against 2 of the 12 strains in this collection that were not highly resistant to gentamicin, the combination of quinupristin-dalfopristin (2 μg/ml) plus gentamicin (5 μg/ml) resulted in killing approaching 3 log
10
CFU/ml. With the exception of doxycycline, inhibitory interactions between quinupristin-dalfopristin and other agents tested against vancomycin-resistant strains of
E. faecium
were uncommon at clinically relevant concentrations.
Comparison of Levofloxacin, Alatrofloxacin, and Vancomycin for Prophylaxis and Treatment of Experimental Foreign-Body-Associated Infection by Methicillin-Resistant
Staphylococcus aureus
Authors: Vaudaux Pierre , Francois Patrice , Bisognano Carmelo , Schrenzel Jacques , Lew Daniel P.
Published: July 2002

The prophylactic and therapeutic activities of two fluoroquinolones, levofloxacin and alatrofloxacin (the
l
-Ala-
l
-Ala prodrug of trovafloxacin), were compared to those of vancomycin in two different experimental models of foreign-body-associated infections caused by methicillin-resistant but quinolone-susceptible
Staphylococcus aureus
(MRSA) isolates. In a guinea pig model of prophylaxis, subcutaneously implanted tissue cages were infected with 10
3
CFU of MRSA, which was a 100% infectious dose in control animals. A single dose of 50 mg of levofloxacin per kg of body weight, administered intraperitoneally 3 h before bacterial challenge, was more efficient than vancomycin for the prevention of infections in tissue cages with MRSA inocula of 10
4
and 10
5
CFU. In a rat model used to evaluate therapy of chronic tissue cage infection caused by MRSA, the efficacies of 7-day high-dose regimens of levofloxacin (100 mg/kg once a day [q.d.] or 50 mg/kg twice a day [b.i.d.]) or alatrofloxacin (50 mg/kg q.d.) were compared to the efficacy of vancomycin (50 mg/kg b.i.d.). Active levels of levofloxacin, trovafloxacin, and vancomycin were continuously present in tissue cage fluid, with the levels exceeding the minimal bactericidal concentrations for MRSA during therapy. The q.d. and b.i.d. regimens of levofloxacin had equivalent activities and were significantly (
P
< 0.05) more active than alatrofloxacin or vancomycin in decreasing the viable counts of MRSA in tissue cage fluids. No quinolone-resistant mutants emerged during therapy with either fluoroquinolone. The mechanisms explaining the inferior activity of alatrofloxacin compared to the activity of levofloxacin against chronic foreign-body-associated infections by MRSA are unknown.
Substrate Specificity of Atrazine Chlorohydrolase and Atrazine-Catabolizing Bacteria
Authors: Seffernick Jennifer L. , Johnson Gilbert , Sadowsky Michael J. , Wackett Lawrence P.
Published: July 2002

Bacterial atrazine catabolism is initiated by the enzyme atrazine chlorohydrolase (AtzA) in
Pseudomonas
sp. strain ADP. Other triazine herbicides are metabolized by bacteria, but the enzymological basis of this is unclear. Here we begin to address this by investigating the catalytic activity of AtzA by using substrate analogs. Purified AtzA from
Pseudomonas
sp. strain ADP catalyzed the hydrolysis of an atrazine analog that was substituted at the chlorine substituent by fluorine. AtzA did not catalyze the hydrolysis of atrazine analogs containing the pseudohalide azido, methoxy, and cyano groups or thiomethyl and amino groups. Atrazine analogs with a chlorine substituent at carbon 2 and
N
-alkyl groups, ranging in size from methyl to
t
-butyl, all underwent dechlorination by AtzA. AtzA catalyzed hydrolytic dechlorination when one nitrogen substituent was alkylated and the other was a free amino group. However, when both amino groups were unalkylated, no reaction occurred. Cell extracts were prepared from five strains capable of atrazine dechlorination and known to contain
atzA
or closely homologous gene sequences:
Pseudomonas
sp. strain ADP,
Rhizobium
strain PATR,
Alcaligenes
strain SG1,
Agrobacterium radiobacter
J14a, and
Ralstonia picketti
D. All showed identical substrate specificity to purified AtzA from
Pseudomonas
sp. strain
ADP
. Cell extracts from
Clavibacter michiganensis
ATZ1, which also contains a gene homologous to
atzA
, were able to transform atrazine analogs containing pseudohalide and thiomethyl groups, in addition to the substrates used by AtzA from
Pseudomonas
sp. strain ADP. This suggests that either (i) another enzyme(s) is present which confers the broader substrate range or (ii) the AtzA itself has a broader substrate range.
Improved Template Preparation for PCR-Based Assays for Detection of Food-Borne Bacterial Pathogens
Authors: Lampel Keith A. , Orlandi Palmer A. , Kornegay Leroy
Published: July 2002

Shigella flexneri
,
Salmonella enterica
serotype Typhimurium, and
Listeria monocytogenes
were applied to FTA filters, and the filters were used directly as templates to demonstrate their sensitivity and applicability in PCR-based detection assays. With pure cultures, the sensitivities of detection by FTA filter-based PCR were 30 to 50 and 200 CFU for the gram-negative enterics and
Listeria
, respectively. Different numbers of
S. flexneri
cells were used in controlled contamination experiments with several different foods (produce, beef, and apple cider). Aliquots from concentrated food washes subsequently spotted onto FTA filters and assayed by PCR gave consistently positive results and detection limits similar to those observed with pure-culture dilutions. This universal method for PCR template preparation from bacterial cells is rapid and highly sensitive and reduces interference from food-associated inhibitors of PCR. In addition, its broad applicability eliminates the need for multiple methods for analysis of food matrices.
Development of a Single-Reaction Multiplex PCR Toxin Typing Assay for
Staphylococcus aureus
Strains
Authors: Sharma Naresh K. , Rees Catherine E. D. , Dodd Christine E. R.
Published: July 2002

We describe here the development of a single-reaction multiplex PCR assay for the enterotoxin genes from
Staphylococcus aureus
that utilizes a universal toxin gene primer in combination with toxin-specific primers to amplify characteristic toxin gene products. In combination with a new DNA purification method, the assay can detect enterotoxin genes A to E from a pure culture within 3 to 4 h. The test was used to characterize a diverse set of environmental
S. aureus
isolates, and a 99% correlation with toxin typing using standard immunological tests was found. The design of the assay allows it to be extended to include both newly characterized and as-yet-unknown toxin genes.
A New System for Three-Dimensional Tracking of Motile Microorganisms
Authors: Thar Roland , Blackburn Nicholas , Kühl Michael
Published: July 2002

A new three-dimensional (3D)-tracking system with optimized dark-field illumination is presented. It allows simultaneous 3D tracking of several free-swimming microorganisms with diameters of >10 μm. Resolution limits and illumination efficiencies for different size classes of microorganisms are treated analytically. First applications for 3D tracking of protists are demonstrated.
Geranic Acid Formation, an Initial Reaction of Anaerobic Monoterpene Metabolism in Denitrifying
Alcaligenes defragrans
Authors: Heyen Udo , Harder Jens
Published: July 2002

Monoterpenes with an unsaturated hydrocarbon structure are mineralized anaerobically by the denitrifying β-proteobacterium
Alcaligenes defragrans
. Organic acids occurring in cells of
A. defragrans
and culture medium were characterized to identify potential products of the monoterpene activation reaction. Geranic acid (
E
,
E
-3,7-dimethyl-2,6-octadienoic acid) accumulated to 0.5 mM in cells grown on α-phellandrene under nitrate limitation. Cell suspensions of
A. defragrans
65Phen synthesized geranic acid in the presence of β-myrcene, α-phellandrene, limonene, or α-pinene. Myrcene yielded the highest transformation rates. The alicyclic acid was consumed by cell suspensions during carbon limitation. Heat-labile substances present in cytosolic extracts catalyzed the formation of geranic acid from myrcene. These results indicated that a novel monoterpene degradation pathway must be present in
A. defragrans.
Relationship between Glycolysis and Exopolysaccharide Biosynthesis in
Lactococcus lactis
Authors: Ramos Ana , Boels Ingeborg C. , de Vos Willem M. , Santos Helena
Published: July 2002

The relationships between glucose metabolism and exopolysaccharide (EPS) production in a
Lactococcus lactis
strain containing the EPS gene cluster (Eps
+
) and in nonproducer strain MG5267 (Eps
−
) were characterized. The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by
31
P nuclear magnetic resonance. The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps
+
strain than in the Eps
−
strain. The precursors of the peptidoglycan pathway, UDP-
N
-acetylglucosamine and UDP-
N
-acetylmuramoyl-pentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps
+
strain, indicating that there is competition between EPS synthesis and cell growth. An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating. Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate. The activities of relevant enzymes, such as phosphoglucose isomerase, α-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains.
13
C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool. This study provided clues for ways to enhance EPS production by genetic manipulation.
Mutations in the Listerial
proB
Gene Leading to Proline Overproduction: Effects on Salt Tolerance and Murine Infection
Authors: Sleator Roy D. , Gahan Cormac G. M. , Hill Colin
Published: July 2002

The observed sensitivity of
Listeria monocytogenes
to the toxic proline analogue
l
-azetidine-2-carboxylic acid (AZ) suggested that proline synthesis in
Listeria
may be regulated by feedback inhibition of γ-glutamyl kinase (GK), the first enzyme of the proline biosynthesis pathway, encoded by the
proB
gene. Taking advantage of the
Epicurian coli
mutator strain XL1-Red, we performed random mutagenesis of the recently described
proBA
operon and generated three independent mutations in the listerial
proB
homologue, leading to proline overproduction and salt tolerance when expressed in an
E. coli
(Δ
proBA
) background. While each of the mutations (located within a conserved 26-amino-acid region of GK) was shown to confer AZ resistance (AZ
r
) on an
L. monocytogenes proBA
mutant, listerial transformants failed to exhibit the salt-tolerant phenotype observed in
E. coli
. Since proline accumulation has previously been linked to the virulence potential of a number of pathogenic bacteria, we analyzed the effect of proline overproduction on
Listeria
pathogenesis. However, our results suggest that as previously described for proline auxotrophy, proline hyperproduction has no apparent impact on the virulence potential of
Listeria
.
Electropositive Filter Membrane as an Alternative for the Elimination of PCR Inhibitors from Sewage and Water Samples
Authors: Queiroz A. P. S. , Santos F. M. , Sassaroli A. , Hársi C. M. , Monezi T. A. , Mehnert D. U.
Published: July 2002

PCR is the best method for the detection of enteric viruses present at low concentrations in environmental samples. However, some organic and inorganic compounds present in these samples can interfere in the reaction. Many of these substances are cytotoxic, too. The ZP60S filter membranes used in addition to fluorpentane treatment are quite efficient for virus concentration and simultaneous elimination of cytotoxicity from environmental samples. In this study, both procedures were used to promote the elimination of reverse transcriptase PCR (RT-PCR) inhibitors from sewage and sewage-polluted creek water. Samples were subjected separately to each of the following procedures: filtration through electropositive filter membranes (ZP60S), organic extraction with Vertrel XF, and filtration through ZP60S followed by organic extraction. Afterwards, aliquots were experimentally inoculated with rotavirus SA-11 RNA and subjected to RT-seminested PCR for amplification of the VP7 gene. Results showed that the ZP60S membranes efficiently eliminated the RT-PCR inhibitors from water samples. The sample processing method was also applied to 31 in natura sewage and creek water samples for detection of naturally occurring rotavirus. A duplex seminested PCR was used for the quick detection of couples of the four rotavirus genotypes (G1 to G4). Eight samples (25.8%) were positive, and rotavirus sequences were not detected in 23 (74.2%). Results were confirmed by direct immunoperoxidase method. In summary, the use of electropositive filter membrane is appropriate for the elimination of substances that can interfere with RT-PCR, obviating additional sample purification methods.
Availability of Glutamate and Arginine during Acid Challenge Determines Cell Density-Dependent Survival Phenotype of
Escherichia coli
Strains
Authors: Cui Shenghui , Meng Jianghong , Bhagwat Arvind A.
Published: July 2002

The cell density-dependent acid sensitivity phenotypes of
Escherichia coli
strains K-12 and O157:H7 were examined with reference to three possible mechanisms of acid resistance. There was no evidence of any diffusible substance released from dead cells which could influence the cell density-dependent acid survival phenotype. Instead, cell density-dependent acid survival phenotype was associated with induction of glutamate- and arginine-decarboxylase acid survival pathways and concomitant availability of glutamate and arginine during acid challenge.
Lyme Disease
Borrelia
spp. in Ticks and Rodents from Northwestern China
Authors: Takada Nobuhiro , Masuzawa Toshiyuki , Ishiguro Fubito , Fujita Hiromi , Kudeken Midori , Mitani Harumi , Fukunaga Masahito , Tsuchiya Kimiyuki , Yano Yasuhiro , Ma Xiao-Hang
Published: July 2002

In May 1999, field surveys of Lyme disease spirochetes were conducted around the Tianshan Mountains in Xinjiang Uygur Autonomous Region in northwestern People's Republic of China.
Ixodes persulcatus
ticks were obtained in a Tianchi Lake valley with primary forest, while the tick fauna was poor in the semidesert or at higher altitudes in this region. Species identities were confirmed by molecular analysis in which an internal transcribed spacer sequence was used. Of 55 adult ticks, 22 (40%) were positive for spirochetes as determined by Barbour-Stoenner-Kelly culture passages. In addition, some rodents, including
Apodemus uralensis
(5 of 14 animals) and
Cricetulus longicaudatus
(the only animal examined), and some immature stages of
I. persulcatus
(4 of 11 ticks) that had fed on
A. uralensis
were positive for spirochetes. Based on 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis and reactivity with monoclonal antibodies, 35 cultures (including double isolation cultures) were identified as
Borrelia garinii
(20 isolates, including 9 Eurasian pattern B isolates and 11 Asian pattern C isolates),
Borrelia afzelii
(10 pattern D isolates), and mixed cultures (5 cultures, including isolates that produced
B. garinii
patterns B and C plus
B. afzelii
pattern D). These findings revealed that Lyme disease pathogens are distributed in the mountainous areas in northwestern China even though it is an arid region, and they also confirmed the specific relationship between
I. persulcatus
and genetic patterns of
Borrelia
spp. on the Asian continent.
Gene Cassette PCR: Sequence-Independent Recovery of Entire Genes from Environmental DNA
Authors: Stokes H. W. , Holmes Andrew J. , Nield Blair S. , Holley Marita P. , Nevalainen K. M. Helena , Mabbutt Bridget C. , Gillings Michael R.
Published: July 2002

The vast majority of bacteria in the environment have yet to be cultured. Consequently, a major proportion of both genetic diversity within known gene families and an unknown number of novel gene families reside in these uncultured organisms. Isolation of these genes is limited by lack of sequence information. Where such sequence data exist, PCR directed at conserved sequence motifs recovers only partial genes. Here we outline a strategy for recovering complete open reading frames from environmental DNA samples. PCR assays were designed to target the 59-base element family of recombination sites that flank gene cassettes associated with integrons. Using such assays, diverse gene cassettes could be amplified from the vast majority of environmental DNA samples tested. These gene cassettes contained complete open reading frames, the majority of which were associated with ribosome binding sites. Novel genes with clear homologies to phosphotransferase, DNA glycosylase, methyl transferase, and thiotransferase genes were identified. However, the majority of amplified gene cassettes contained open reading frames with no identifiable homologues in databases. Accumulation analysis of the gene cassettes amplified from soil samples showed no signs of saturation, and soil samples taken at 1-m intervals along transects demonstrated different amplification profiles. Taken together, the genetic novelty, steep accumulation curves, and spatial heterogeneity of genes recovered show that this method taps into a vast pool of unexploited genetic diversity. The success of this approach indicates that mobile gene cassettes and, by inference, integrons are widespread in natural environments and are likely to contribute significantly to bacterial diversity.
Detection of the
isiA
Gene across Cyanobacterial Strains: Potential for Probing Iron Deficiency
Authors: Geiß U. , Vinnemeier J. , Kunert A. , Lindner I. , Gemmer B. , Lorenz M. , Hagemann M. , Schoor A.
Published: July 2002

The use of
isiA
expression to monitor the iron status of cyanobacteria was investigated. Studies of laboratory cultures of the cyanobacterium
Synechocystis
sp. strain PCC 6803 showed that
isiA
expression is dependent on the organism's response to iron deficiency;
isiA
expression starts as soon as a decline in the rate of growth begins.
isiA
expression is switched on at concentrations of iron citrate of less than 0.7 μM. A PCR method was developed for the specific amplification of the iron-regulated
isiA
gene from a variety of cyanobacteria. After we developed degenerate primers, 15 new internal
isiA
fragments (840 bp) were amplified, cloned, and sequenced from strains obtained from algal collections, from new isolates, and from enriched field samples. Furthermore,
isiA
expression could be detected by means of reverse transcription-PCR when enriched field samples were exposed to restricted iron availability. These results imply that determining the level of iron-regulated
isiA
expression can serve to indicate iron deficiency in cyanobacterial samples of differing origins from the field.
Enterotoxigenic Potential ofStaphylococcus intermedius
Authors: Becker Karsten , Keller Birgit , von Eiff Christof , Brück Michaela , Lubritz Gabriele , Etienne Jerome , Peters Georg
Published: July 2002

Staphylococcal food poisoning (SFP) caused by enterotoxigenic staphylococci is one of the main food-borne diseases. In contrast toStaphylococcus aureus, a systematic screening for the enterotoxins has not yet been performed on the genomic level for the coagulase-positive speciesS.intermedius. Therefore, the enterotoxigenic potential of 281 different veterinary (canine,n= 247; equine,n= 23; feline,n= 9; other,n= 2) and 11 human isolates ofS.intermediuswas tested by using a multiplex PCR DNA-enzyme immunoassay system targeting the staphylococcal enterotoxin genessea,seb,sec,sed, andsee. Molecular results were compared by in vitro testing of enterotoxin production by two immunoassays. A total of 33 (11.3%)S.intermediusisolates, including 31 (12.6%) canine isolates, 1 equine isolate, and 1 human isolate, tested positive for thesecgene. In vitro production of the respective enterotoxins was detected in 30 (90.9%) of these isolates by using immunological tests. In contrast, none of 65 veterinary specimen-derived isolates additionally tested and comprising 13 (sub)species of coagulase-negative staphylococci were found to be enterotoxigenic. This study shows on both molecular and immunological levels that a substantial number ofS.intermediusisolates harbor the potential for enterotoxin production. Since evidence for noninvasive zoonotic transmission ofS.intermediusfrom animal hosts to humans has been documented, an enterotoxigenic role of this microorganism in SFP via contamination of food products may be assumed.
Role of β-Oxidation Enzymes in γ-Decalactone Production by the Yeast
Yarrowia lipolytica
Authors: Waché Yves , Aguedo Mario , Choquet Armelle , Gatfield Ian L. , Nicaud Jean-Marc , Belin Jean-Marc
Published: July 2002

Some microorganisms can transform methyl ricinoleate into γ-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C
18
) to the C
10
precursor of γ-decalactone, (ii) accumulation of other lactones (3-hydroxy-γ-decalactone and 2- and 3-decen-4-olide), and (iii) γ-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the
POX1
through
POX5
genes) in
Yarrowia lipolytica
in lactone accumulation and γ-decalactone reconsumption in
POX
mutants. Mutants with no acyl-CoA oxidase activity could not reconsume γ-decalactone, and mutants with a disruption of
pox3
, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-γ-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, β-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the β-oxidation flux. We also identified mutant strains that produced 26 times more γ-decalactone than the wild-type parents.
Application of Digital Image Analysis and Flow Cytometry To Enumerate Marine Viruses Stained with SYBR Gold
Authors: Chen Feng , Lu Jing-rang , Binder Brian J. , Liu Ying-chun , Hodson Robert E.
Published: July 2002

A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.
Molecular Characterization and Identification of
Bacillus clausii
Strains Marketed for Use in Oral Bacteriotherapy
Authors: Senesi Sonia , Celandroni Francesco , Tavanti Arianna , Ghelardi Emilia
Published: July 2002

A substantial number of
Bacillus
species have been marketed for use in oral bacteriotherapy because of their purported ability to prevent or treat various gastrointestinal disorders. Recently, some of the
Bacillus
strains in Enterogermina, which is made up of aqueous suspensions of viable
Bacillus
spores, have been partially characterized and aligned with members of the
Bacillus alcalophilus
subgroup rather than with
Bacillus subtilis
, as previously reported. With a view toward verifying the original taxonomic position of the Enterogermina strains, we catalogued both phenotypic and genotypic traits exhibited by the four
Bacillus
strains isolated from the spore mixtures found in original commercial preparations dated 1975 and 1984 and commercial preparations now being propagated industrially. Analyses of physiological and biochemical traits, complete 16S rRNA gene sequences, DNA-DNA reassociation, tRNA intergenic spacer length polymorphism, single-strand conformation polymorphism of PCR-amplified spacer regions of tRNA genes, and randomly amplified polymorphic DNA led to the finding that all of the Enterogermina strains belong to a unique genospecies, which is unequivocally identified as the alkalitolerant species
Bacillus clausii
. Moreover, we provide evidence that in contrast to several reference strains of
B. clausii
, the strains constituting Enterogermina are characterized by a notable low level of intraspecific genome diversity and that each strain has remained the same for the last 25 years.
Congruent Phylogenies of Most Common Small-Subunit rRNA and Dissimilatory Sulfite Reductase Gene Sequences Retrieved from Estuarine Sediments
Authors: Joulian Catherine , Ramsing Niels B. , Ingvorsen Kjeld
Published: July 2002

The diversity of sulfate-reducing bacteria (SRB) in brackish sediment was investigated using small-subunit rRNA and dissimilatory sulfite reductase (DSR) gene clone libraries and cultivation. The phylogenetic affiliation of the most commonly retrieved clones for both genes was strikingly similar and produced
Desulfosarcina variabilis
-like sequences from the inoculum but
Desulfomicrobium baculatum
-like sequences from a high dilution in natural media. Related organisms were subsequently cultivated from the site. PCR bias appear to be limited (or very similar) for the two primersets and target genes. However, the DSR primers showed a much higher phylogenetic specificity. DSR gene analysis is thus a promising and specific approach for investigating SRB diversity in complex habitats.
Three Glycoproteins with Antimutagenic Activity Identified in
Lactobacillus plantarum
KLAB21
Authors: Rhee Chang-Ho , Park Heui-Dong
Published: July 2002

Antimutagenic substances were purified from a culture supernatant of
Lactobacillus plantarum
KLAB21 cells isolated from kimchi, a Korean traditional fermented vegetable, and their characteristics were investigated. The antimutagenic substances were separated into two fractions by DEAE-cellulose ion-exchange column chromatography, which were designated the R1 and R2 fractions. The R1 fraction was then divided into two fractions again by Sephadex G200 gel filtration chromatography, and the fractions were designated R1-1 and R1-2. All three fractions were further purified using a Sepharose CL-6B gel filtration column. All the purified fractions were successfully stained with fuchsin as well as Coomassie brilliant blue, suggesting that they are glycoproteins. The purified fractions were confirmed to possess antimutagenic activity against
N
-methyl-
N
′-nitro-
N
-nitrosoguanidine on
Salmonella enterica
serovar Typhimurium TA100 cells. Their molecular masses were determined to be 16 (R1-1), 11 (R1-2), and 14 (R2) kDa on the Sepharose CL-6B column. Total sugar contents were 8.4% (R1-1), 7.3% (R1-2), and 9.4% (R2). The amino acid compositions of the fractions were different from each other; the major amino acids were glutamic acid (21.5%) and phenylalanine (17.1%) in the R1-1 fraction and glycine (41.3%) in the R1-2 fraction, but valine (31%) and phenylalanine (22.6%) were the major amino acids in the R2 fraction.
Quantitative Analysis of the Relative Transcript Levels of ABC Transporter
Atr
Genes in
Aspergillus nidulans
by Real-Time Reverse Transcription-PCR Assay
Authors: Semighini Camile Pizeta , Marins Mozart , Goldman Maria Helena S. , Goldman Gustavo Henrique
Published: July 2002

The development of assays for quantitative analysis of the relative transcript levels of ABC transporter genes by real-time reverse transcription-PCR (RT-PCR) might provide important information about multidrug resistance in filamentous fungi. Here, we evaluate the potential of real-time RT-PCR to quantify the relative transcript levels of ABC transporter
Atr
genes from
Aspergillus nidulans
. The
AtrA
to
AtrD
genes showed different and higher levels in the presence of structurally unrelated drugs, such as camptothecin, imazalil, itraconazole, hygromycin, and 4-nitroquinoline oxide. We also verified the relative transcript levels of the
Atr
genes in the
A. nidulans
imazalil-resistant mutants. These genes displayed a very complex pattern in different
ima
genetic backgrounds. The
imaB
mutant has higher basal transcript levels of
AtrB
and -
D
than those of the wild-type strain. The levels of these two genes are comparable when the
imaB
mutant is grown in the presence and absence of imazalil. The
imaC
, -
D
, and -
H
mutants have higher basal levels of
AtrA
than that of the wild type. The same behavior is observed for the relative transcript levels of
AtrB
in the
imaG
mutant background.
Clonal Analysis of the Human B-Cell Repertoire Using a Heteromobility Assay
Authors: Tewari Deepanker
Published: July 2002

A heteromobility duplex tracking assay was developed to analyze B-cell clonality. The assay was based on the genetic variability of B-cell immunoglobulin (Ig) sequences. Binding of amplified (Ig) sequences to a single-stranded radiolabeled Ig DNA probe resulted in the formation of heteroduplexes. The mobilities of these heteroduplexes helped to distinguish clonal B cells.