bluuebunny/crossref_metadata_2025 huggingface dataset:
Cumulative Effect of Amino Acid Replacements Results in Enhanced Thermostability of Potato Type L α-Glucan Phosphorylase
Authors: Yanase Michiyo , Takata Hiroki , Fujii Kazutoshi , Takaha Takeshi , Kuriki Takashi
Published: September 2005

The thermostability of potato type L α-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations—Phe39→Leu (F39L), Asn135→Ser (N135S), and Thr706→Ile (T706I)—by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60°C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65°C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.
Novel Chemoautotrophic Endosymbiosis between a Member of the
Epsilonproteobacteria
and the Hydrothermal-Vent Gastropod
Alviniconcha
aff.
hessleri
(Gastropoda: Provannidae) from the Indian Ocean
Authors: Suzuki Yohey , Sasaki Takenori , Suzuki Masae , Nogi Yuichi , Miwa Tetsuya , Takai Ken , Nealson Kenneth H. , Horikoshi Koki
Published: September 2005

The hydrothermal-vent gastropod
Alviniconcha
aff.
hessleri
from the Kairei hydrothermal field on the Central Indian Ridge houses bacterium-like cells internally in its greatly enlarged gill. A single 16S rRNA gene sequence was obtained from the DNA extract of the gill, and phylogenetic analysis placed the source organism within a lineage of the epsilon subdivision of the
Proteobacteria
. Fluorescence in situ hybridization analysis with an oligonucleotide probe targeting the specific epsilonproteobacterial subgroup showed the bacterium densely colonizing the gill filaments. Carbon isotopic homogeneity among the gastropod tissue parts, regardless of the abundance of the endosymbiont cells, suggests that the carbon isotopic composition of the endosymbiont biomass is approximately the same as that of the gastropod. Compound-specific carbon isotopic analysis revealed that fatty acids from the gastropod tissues are all
13
C enriched relative to the gastropod biomass and that the monounsaturated C
16
fatty acid that originates from the endosymbiont is as
13
C enriched relative to the gastropod biomass as that of the epsilonproteobacterial cultures grown under chemoautotrophic conditions. This fractionation pattern is most likely due to chemoautotrophy based on the reductive tricarboxylic-acid (rTCA) cycle and subsequent fatty acid biosynthesis from
13
C-enriched acetyl coenzyme A. Enzymatic characterization revealed evident activity of several key enzymes of the rTCA cycle, as well as the absence of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in the gill tissue. The results from anatomic, molecular phylogenetic, bulk and compound-specific carbon isotopic, and enzymatic analyses all support the inference that a novel nutritional strategy relying on chemoautotrophy in the epsilonproteobacterial endosymbiont is utilized by the hydrothermal-vent gastropod from the Indian Ocean. The discrepancies between the data of the present study and those of previous ones for
Alviniconcha
gastropods from the Pacific Ocean imply that at least two lineages of chemoautotrophic bacteria, phylogenetically distinct at the subdivision level, occur as the primary endosymbiont in one host animal type.
Design and Evaluation of 16S rRNA-Targeted Peptide Nucleic Acid Probes for Whole-Cell Detection of Members of the Genus
Listeria
Authors: Brehm-Stecher Byron F. , Hyldig-Nielsen Jens J. , Johnson Eric A.
Published: September 2005

Six fluorescein-labeled peptide nucleic acid oligomers targeting
Listeria
-specific sequences on the 16S ribosomal subunit were evaluated for their abilities to hybridize to whole cells by fluorescence in situ hybridization (FISH). Four of these probes yielded weak or no fluorescent signals after hybridization and were not investigated further. The remaining two FISH-compatible probes, LisUn-3 and LisUn-11, were evaluated for their reactivities against 22
Listeria
strains and 17 other bacterial strains belonging to 10 closely related genera. Hybridization with BacUni-1, a domain-specific eubacterial probe, was used as a positive control for target accessibility in both
Listeria
spp. and nontarget cells. RNase T1 treatment of select cell types was used to confirm that positive fluorescence responses were rRNA dependent and to examine the extent of nonspecific staining of nontarget cells. Both LisUn-3 and LisUn-11 yielded rapid, bright, and genus-specific hybridizations at probe concentrations of approximately 100 pmol ml
−1
. LisUn-11 was the brightest probe and stained all six
Listeria
species. LisUn-3 hybridized with all
Listeria
spp. except for
L. grayi
, for which it had two mismatched bases. A simple ethanolic fixation yielded superior results with
Listeria
spp. compared to fixation in 10% buffered formalin and was applicable to all cell types studied. This study highlights the advantages of peptide nucleic acid probes for FISH-based detection of gram-positive bacteria and provides new tools for the rapid detection of
Listeria
spp. These probes may be useful for the routine monitoring of food production environments in support of efforts to control
L. monocytogenes
.
Interaction of
Pasteurella multocida
with Free-Living Amoebae
Authors: Hundt Matthew J. , Ruffolo Carmel G.
Published: September 2005

Pasteurella multocida
is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between
P. multocida
and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing
P. multocida
strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of
P. multocida
within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between
P. multocida
and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between
P. multocida
and host cells. Free-living amoebae are ubiquitous within water and soil environments, and
P. multocida
has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between
P. multocida
and amoebae may occur within the natural environment.
Generation and Phenotypic Characterization of
Aspergillus nidulans
Methylisocitrate Lyase Deletion Mutants: Methylisocitrate Inhibits Growth and Conidiation
Authors: Brock Matthias
Published: September 2005

Propionate is a very abundant carbon source in soil, and many microorganisms are able to use this as the sole carbon source. Nevertheless, propionate not only serves as a carbon source for filamentous fungi but also acts as a preservative when added to glucose containing media. To solve this contradiction between carbon source and preservative effect, propionate metabolism of
Aspergillus nidulans
was studied and revealed the methylcitrate cycle as the responsible pathway. Methylisocitrate lyase is one of the key enzymes of that cycle. It catalyzes the cleavage of methylisocitrate into succinate and pyruvate and completes the α-oxidation of propionate. Previously, methylisocitrate lyase was shown to be highly specific for the substrate (2
R
,3
S
)-2-methylisocitrate. Here, the identification of the genomic sequence of the corresponding gene and the generation of deletion mutants is reported. Deletion mutants did not grow on propionate as sole carbon and energy source and were severely inhibited during growth on alternative carbon sources, when propionate was present. The strongest inhibitory effect was observed, when glycerol was the main carbon source, followed by glucose and acetate. In addition, asexual conidiation was strongly impaired in the presence of propionate. These effects might be caused by competitive inhibition of the NADP-dependent isocitrate dehydrogenase, because the

K
i

of (2
R
,3
S
)-2-methylisocitrate, the product of the methylcitrate cycle, on NADP-dependent isocitrate dehydrogenase was determined as 1.55 μM. Other isomers had no effect on enzymatic activity. Therefore, methylisocitrate was identified as a potential toxic compound for cellular metabolism.
Mutations of Toluene-4-Monooxygenase That Alter Regiospecificity of Indole Oxidation and Lead to Production of Novel Indigoid Pigments
Authors: McClay Kevin , Boss Corinne , Keresztes Ivan , Steffan Robert J.
Published: September 2005

Broad-substrate-range monooygenase enzymes, including toluene-4-monooxygenase (T4MO), can catalyze the oxidation of indole. The indole oxidation products can then condense to form the industrially important dye indigo. Site-directed mutagenesis of T4MO resulted in the creation of T4MO isoforms with altered pigment production phenotypes. High-pressure liquid chromatography, thin-layer chromatography, and nuclear magnetic resonance analysis of the indole oxidation products generated by the mutant T4MO isoforms revealed that the phenotypic differences were primarily due to changes in the regiospecificity of indole oxidation. Most of the mutations described in this study changed the ratio of the primary indole oxidation products formed (indoxyl, 2-oxindole, and isatin), but some mutations, particularly those involving amino acid G103 of
tmoA
, allowed for the formation of additional products, including 7-hydroxyindole and novel indigoid pigments. For example, mutant G103L converted 17% of added indole to 7-hydroxyindole and 29% to indigoid pigments including indigo and indirubin and two other structurally related pigments. The double mutant G103L:A107G converted 47% of indole to 7-hydroxyindole, but no detectable indigoid pigments were formed, similar to the product distribution observed with the toluene-2-monooxygenase (T2MO) of
Burkholderia cepacia
G4. These results demonstrate that modification of the
tmoA
active site can change the products produced by the enzyme and lead to the production of novel pigments and other indole oxidation products with potential commercial and medicinal utility.
Spatial Organization of Dual-Species Bacterial Aggregates on Leaf Surfaces
Authors: Monier J.-M. , Lindow S. E.
Published: September 2005

The spatial organization of cells within bacterial aggregates on leaf surfaces was determined for pair-wise mixtures of three different bacterial species commonly found on leaves,
Pseudomonas syringae
,
Pantoea agglomerans
, and
Pseudomonas fluorescens
. Cells were coinoculated onto bean plants and allowed to grow under moist conditions, and the resulting aggregates were examined in situ by epifluorescence microscopy. Each bacterial strain could be localized because it expressed either the green or the cyan fluorescent protein constitutively, and the viability of individual cells was assessed by propidium iodide staining. Each pair of bacterial strains that was coinoculated onto leaves formed mixed aggregates. The degree of segregation of cells in mixed aggregates differed between the different coinoculated pairs of strains and was higher in mixtures of
P. fluorescens
A506 and
P. agglomerans
299R and mixtures of
P. syringae
B728a and
P. agglomerans
299R than in mixtures of two isogenic strains of
P. agglomerans
299R. The fractions of the total cell population that were dead in mixed and monospecific aggregates of a
gfp
-marked strain of
P. agglomerans
299R and a
cfp
-marked strain of
P. agglomerans
299R, or of
P. fluorescens
A506 and
P. agglomerans
299R, were similar. However, the proportion of dead cells in mixed aggregates of
P. syringae
B728a and
P. agglomerans
299R was significantly higher (13.2% ± 8.2%) than that in monospecific aggregates of these two strains (1.6% ± 0.7%), and it increased over time. While dead cells in such mixed aggregates were preferentially found at the interface between clusters of cells of these strains, cells of these two strains located at the interface did not exhibit equal probabilities of mortality. After 9 days of incubation, about 77% of the
P. agglomerans
299R cells located at the interface were dead, while only about 24% of the
P. syringae
B728a cells were dead. The relevance of our results to understanding bacterial interactions on leaf surfaces and the implications for biological control of pathogenic and other deleterious microorganisms is discussed.
Whole-Genome Shotgun Optical Mapping of
Rhodospirillum rubrum
Authors: Reslewic Susan , Zhou Shiguo , Place Mike , Zhang Yaoping , Briska Adam , Goldstein Steve , Churas Chris , Runnheim Rod , Forrest Dan , Lim Alex , Lapidus Alla , Han Cliff S. , Roberts Gary P. , Schwartz David C.
Published: September 2005

Rhodospirillum rubrum
is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of
R. rubrum
strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a “molecular cytogenetics” approach to solving problems in genomic analysis.
rRNA Sequence-Based Scanning Electron Microscopic Detection of Bacteria
Authors: Kenzaka Takehiko , Ishidoshiro Ai , Yamaguchi Nobuyasu , Tani Katsuji , Nasu Masao
Published: September 2005

A new scanning electron microscopic method was developed for gaining both phylogenetic and morphological information about target microbes using in situ hybridization with rRNA-targeted oligonucleotide probes (SEM-ISH). Target cells were hybridized with oligonucleotide probes after gold labeling. Gold enhancement was used for amplification of probe signals from hybridized cells. The hybridized cells released a strong backscatter electron signal due to accumulation of gold atoms inside cells. SEM-ISH was applied to analyze bacterial community composition in freshwater samples, and bacterial cell counts determined by SEM-ISH with rRNA-targeted probes for major phyla within the domain
Bacteria
were highly correlated to those by fluorescent in situ hybridization (FISH). The bacterial composition on surface of river sediment particles before and after cell dispersion treatment by sonication was successfully revealed by SEM-ISH. Direct enumeration of bacterial cells on the surface of sonicated sediment particles by SEM-ISH demonstrated that members of
Cytophaga-Flavobacterium
existed tightly on the surface of particles. SEM-ISH allows defining the number and distribution of phylogenetically defined cells adherent to material surfaces, which is difficult in FISH, and it gives new insight into electron microscopic studies of microorganisms in their natural environment.
Complexation of Uranium by Cells and S-Layer Sheets of
Bacillus sphaericus
JG-A12
Authors: Merroun Mohamed L. , Raff Johannes , Rossberg André , Hennig Christoph , Reich Tobias , Selenska-Pobell Sonja
Published: September 2005

Bacillus sphaericus
JG-A12 is a natural isolate recovered from a uranium mining waste pile near the town of Johanngeorgenstadt in Saxony, Germany. The cells of this strain are enveloped by a highly ordered crystalline proteinaceous surface layer (S-layer) possessing an ability to bind uranium and other heavy metals. Purified and recrystallized S-layer proteins were shown to be phosphorylated by phosphoprotein-specific staining, inductive coupled plasma mass spectrometry analysis, and a colorimetric method. We used extended X-ray absorption fine-structure (EXAFS) spectroscopy to determine the structural parameters of the uranium complexes formed by purified and recrystallized S-layer sheets of
B. sphaericus
JG-A12. In addition, we investigated the complexation of uranium by the vegetative bacterial cells. The EXAFS analysis demonstrated that in all samples studied, the U(VI) is coordinated to carboxyl groups in a bidentate fashion with an average distance between the U atom and the C atom of 2.88 ± 0.02 Å and to phosphate groups in a monodentate fashion with an average distance between the U atom and the P atom of 3.62 ± 0.02 Å. Transmission electron microscopy showed that the uranium accumulated by the cells of this strain is located in dense deposits at the cell surface.
Fungal Community Analysis by Large-Scale Sequencing of Environmental Samples
Authors: O'Brien Heath E. , Parrent Jeri Lynn , Jackson Jason A. , Moncalvo Jean-Marc , Vilgalys Rytas
Published: September 2005

Fungi are an important and diverse component of soil communities, but these communities have proven difficult to study in conventional biotic surveys. We evaluated soil fungal diversity at two sites in a temperate forest using direct isolation of small-subunit and internal transcribed spacer (ITS) rRNA genes by PCR and high-throughput sequencing of cloned fragments. We identified 412 sequence types from 863 fungal ITS sequences, as well as 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were present in both the ITS and small-subunit libraries, while members of other fungal phyla were recovered at much lower frequencies. Many sequences closely matched sequences from mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among samples from different soil depths, with mycorrhizal species predominating deeper in the soil profile and saprophytic species predominating in the litter layer. Richness was consistently lowest in the deepest soil horizon samples. Comparable levels of fungal richness have been observed following traditional specimen-based collecting and culturing surveys, but only after much more extensive sampling. The high rate at which new sequence types were recovered even after sampling 863 fungal ITS sequences and the dominance of fungi in our libraries relative to other eukaryotes suggest that the abundance and diversity of fungi in forest soils may be much higher than previously hypothesized.
Abundances, Identity, and Growth State of Actinobacteria in Mountain Lakes of Different UV Transparency
Authors: Warnecke Falk , Sommaruga Ruben , Sekar Raju , Hofer Julia S. , Pernthaler Jakob
Published: September 2005

The occurrence, identity, and activity of microbes from the class
Actinobacteria
was studied in the surface waters of 10 oligo- to mesotrophic mountain lakes located between 913 m and 2,799 m above sea level. Oligonucleotide probes were designed to distinguish between individual lineages within this group by means of fluorescence in situ hybridization (FISH). Bacteria of a single phylogenetic lineage (acI) represented >90% of all
Actinobacteria
in the studied lakes, and they constituted up to 70% of the total bacterial abundances. In the subset of eight lakes situated above the treeline, the community contribution of bacteria from the acI lineage was significantly correlated with the ambient levels of solar UV radiation (UV transparency,
r
2
= 0.72;
P
< 0.01). Three distinct genotypic subpopulations were distinguished within acI that constituted varying fractions of all
Actinobacteria
in the different lakes. The abundance of growing actinobacterial cells was estimated by FISH and immunocytochemical detection of bromodeoxyuridine (BrdU) incorporation into de novo-synthesized DNA. The percentages of
Actinobacteria
with visible DNA synthesis approximately corresponded to the average percentages of BrdU-positive cells in the total assemblages. 
Actinobacteria
from different subclades of the acI lineage, therefore, constituted an important autochthonous element of the aquatic microbial communities in many of the studied lakes, potentially also due to their higher UV resistance.
Survival of
Campylobacter jejuni
in Waterborne Protozoa
Authors: Snelling W. J. , McKenna J. P. , Lecky D. M. , Dooley J. S. G.
Published: September 2005

The failure to reduce the
Campylobacter
contamination of intensively reared poultry may be partially due to
Campylobacter
resisting disinfection in water after their internalization by waterborne protozoa.
Campylobacter jejuni
and a variety of waterborne protozoa, including ciliates, flagellates, and alveolates, were detected in the drinking water of intensively reared poultry by a combination of culture and molecular techniques. An in vitro assay showed that
C. jejuni
remained viable when internalized by
Tetrahymena pyriformis
and
Acanthamoeba castellanii
for significantly longer (up to 36 h) than when they were in purely a planktonic state. The internalized
Campylobacter
were also significantly more resistant to disinfection than planktonic organisms. Collectively, our results strongly suggest that protozoa in broiler drinking water systems can delay the decline of
Campylobacter
viability and increase
Campylobacter
disinfection resistance, thus increasing the potential of
Campylobacter
to colonize broilers.
Disintegration of
Staphylococcus epidermidis
Biofilms under Glucose-Limiting Conditions Depends on the Activity of the Alternative Sigma Factor σ
B
Authors: Jäger Sebastian , Mack Dietrich , Rohde Holger , Horstkotte Matthias A. , Knobloch Johannes K.-M.
Published: September 2005

To evaluate the role of the polysaccharide intercellular adhesin as an energy-storage molecule, we investigated the effect of nutrient limitation on
S. epidermidis
biofilms. The stability of established biofilms depends on σ
B
activity; however, the slow decay of biofilms under conditions of nutrient limitation reveal its use as an energy-storage molecule to be unlikely.
Inactivation of Spores of
Bacillus anthracis
Sterne,
Bacillus cereus
, and
Bacillus thuringiensis
subsp.
israelensis
by Chlorination
Authors: Rice E. W. , Adcock N. J. , Sivaganesan M. , Rose L. J.
Published: September 2005

Three species of
Bacillus
were evaluated as potential surrogates for
Bacillus anthracis
for determining the sporicidal activity of chlorination as commonly used in drinking water treatment. Spores of
Bacillus thuringiensis
subsp.
israelensis
were found to be an appropriate surrogate for spores of
B. anthracis
for use in chlorine inactivation studies.
Biosensor Detection of Botulinum Toxoid A and Staphylococcal Enterotoxin B in Food
Authors: Sapsford Kim E. , Taitt Chris R. , Loo Nicole , Ligler Frances S.
Published: September 2005

Immunoassays were developed for the simultaneous detection of staphylococcal enterotoxin B and botulinum toxoid A in buffer, with limits of detection of 0.1 ng/ml and 20 ng/ml, respectively. The toxins were also spiked and measured in a variety of food samples, including canned tomatoes, sweet corn, green beans, mushrooms, and tuna.
Identification of DNA Sequences Specific for
Vibrio vulnificus
Biotype 2 Strains by Suppression Subtractive Hybridization
Authors: Lee Chung-Te , Amaro Carmen , Sanjuán Eva , Hor Lien-I
Published: September 2005

Vibrio vulnificus
can be divided into three biotypes, and only biotype 2, which is further divided into serovars, contains eel-virulent strains. We compared the genomic DNA of a biotype 2 serovar E isolate (tester) with the genomic DNAs of three biotype 1 strains by suppression subtractive hybridization and then tested the distribution of the tester-specific DNA sequences in a wide collection of bacterial strains. In this way we identified three plasmid-borne DNA sequences that were specific for biotype 2 strains irrespective of the serovar and three chromosomal DNA sequences that were specific for serovar E biotype 2 strains. These sequences have potential for use in the diagnosis of eel vibriosis caused by
V. vulnificus
and in the detection of biotype 2 serovar E strains.
Isolation of Multidrug-Resistant
Stenotrophomonas maltophilia
from Cultured Yellowtail (
Seriola quinqueradiata
) from a Marine Fish Farm
Authors: Furushita Manabu , Okamoto Akira , Maeda Toshimichi , Ohta Michio , Shiba Tsuneo
Published: September 2005

Six strains of multidrug-resistant
Stenotrophomonas maltophilia
were isolated from cultured yellowtail. The strains were divided into two clusters based on the 16S rRNA genes, and all of them contained L1 metallo-β-lactamase and L2 β-lactamase genes. Differences in the intercluster divergence between the lactamase genes suggest that horizontal transfer of the genes occurred.
Surface Attachment of
Listeria monocytogenes
Is Induced by Sublethal Concentrations of Alcohol at Low Temperatures
Authors: Gravesen Anne , Lekkas Charidimos , Knøchel Susanne
Published: September 2005

Sublethal concentrations of ethanol or isopropanol increased attachment of
Listeria monocytogenes
at 10, 20, or 30°C; no induction occurred at 37°C. The alcohol induction phenotype was retained in
sigB
and
cesRK
mutants; however, the degree of induction was affected. These results suggest that alcohol may contribute to the persistence of
L. monocytogenes
.
Biosynthesis of Natural Flavanones in
Saccharomyces cerevisiae
Authors: Yan Yajun , Kohli Abhijeet , Koffas Mattheos A. G.
Published: September 2005

A four-step flavanone biosynthetic pathway was constructed and introduced into
Saccharomyces cerevisiae
. The recombinant yeast strain was fed with phenylpropanoid acids and produced the flavanones naringenin and pinocembrin 62 and 22 times more efficiently compared to previously reported recombinant prokaryotic strains. Microbial biosynthesis of the flavanone eriodictyol was also achieved.
Comparative Inactivation of Adenovirus Serotypes by UV Light Disinfection
Authors: Nwachuku Nena , Gerba Charles P. , Oswald Amy , Mashadi Faezeh D.
Published: September 2005

The results of this study confirm that adenoviruses are the most resistant enteric viruses to inactivation by UV light and that adenovirus 40 appears to be the most resistant. The effect of freeze-thawing and storage in water may affect the sensitivity of some adenoviruses to inactivation by UV light.
Nitrogenase Switch-Off by Ammonium Ions in
Azospirillum brasilense
Requires the GlnB Nitrogen Signal-Transducing Protein
Authors: Klassen Giseli , Souza Emanuel M. , Yates M. Geoffrey , Rigo Liu Un , Costa Roberta M. , Inaba Juliana , Pedrosa Fábio O.
Published: September 2005

Nitrogenase activity in several diazotrophs is switched off by ammonium and reactivated after consumption. The signaling pathway to this system in
Azospirillum brasilense
is not understood. We show that ammonium-dependent switch-off through ADP-ribosylation of Fe protein was partial in a
glnB
mutant of
A. brasilense
but absent in a
glnB glnZ
double mutant. Triggering of inactivation by anaerobic conditions was not affected in either mutant. The results suggest that
glnB
is necessary for full ammonium-dependent nitrogenase switch-off in
A. brasilense
.
Extracellular Protease of
Pseudomonas fluorescens
CHA0, a Biocontrol Factor with Activity against the Root-Knot Nematode
Meloidogyne incognita
Authors: Siddiqui Imran Ali , Haas Dieter , Heeb Stephan
Published: September 2005

In
Pseudomonas fluorescens
CHA0, mutation of the GacA-controlled
aprA
gene (encoding the major extracellular protease) or the
gacA
regulatory gene resulted in reduced biocontrol activity against the root-knot nematode
Meloidogyne incognita
during tomato and soybean infection. Culture supernatants of strain CHA0 inhibited egg hatching and induced mortality of
M. incognita
juveniles more strongly than did supernatants of
aprA
and
gacA
mutants, suggesting that AprA protease contributes to biocontrol.
Characterization of a Multicopper Oxidase Gene from
Staphylococcus aureus
Authors: Sitthisak S. , Howieson K. , Amezola C. , Jayaswal R. K.
Published: September 2005

A multicopper oxidase gene from
Staphylococcus aureus
was cloned and overexpressed. Purified recombinant multicopper oxidase oxidized the substrate 3,3′-dimethoxybenzidine in the presence of copper. Disruption of
mco
showed copper sensitivity and H
2
O
2
resistance, suggesting roles for
mco
in copper homeostasis and oxidative stress response. Northern blot analysis showed copper-induced
mco
transcription.
Genetic Loci of Major Antigenic Protein Genes ofEdwardsiella tarda
Authors: Verjan Noel , Hirono Ikuo , Aoki Takashi
Published: September 2005

Seven antigenic proteins ofEdwardsiella tardawere identified by using a rabbit polyclonal antiserum. Four of these proteins also reacted with a Japanese flounder antiserum. The amino acid sequences had identity to lipoproteins, periplasmic proteins, and exported and secreted proteins with roles in transport of metabolites across the cell membrane, stress response, and motility. These genes and their products are useful for developing DNA or recombinant subunit vaccines to control edwardsiellosis.
Release of Ca
2+
from the Endoplasmic Reticulum Contributes to Ca
2+
Signaling in
Dictyostelium discoideum
Authors: Wilczynska Zofia , Happle Kathrin , Müller-Taubenberger Annette , Schlatterer Christina , Malchow Dieter , Fisher Paul R.
Published: September 2005

Ca
2+
responses to two chemoattractants, folate and cyclic AMP (cAMP), were assayed in
Dictyostelium D. discoideum
mutants deficient in one or both of two abundant Ca
2+
-binding proteins of the endoplasmic reticulum (ER), calreticulin and calnexin. Mutants deficient in either or both proteins exhibited enhanced cytosolic Ca
2+
responses to both attractants. Not only were the mutant responses greater in amplitude, but they also exhibited earlier onsets, faster rise rates, earlier peaks, and faster fall rates. Correlations among these kinetic parameters and the response amplitudes suggested that key events in the Ca
2+
response are autoregulated by the magnitude of the response itself, i.e., by cytosolic Ca
2+
levels. This autoregulation was sufficient to explain the altered kinetics of the mutant responses: larger responses are faster in both mutant and wild-type cells in response to both folate (vegetative cells) and cAMP (differentiated cells). Searches of the predicted
D. discoideum
proteome revealed three putative Ca
2+
pumps and four putative Ca
2+
channels. All but one contained sequence motifs for Ca
2+
- or calmodulin-binding sites, consistent with Ca
2+
signals being autoregulatory. Although cytosolic Ca
2+
responses in the calnexin and calreticulin mutants are enhanced, the influx of Ca
2+
from the extracellular medium into the mutant cells was smaller. Compared to wild-type cells, Ca
2+
release from the ER in the mutants thus contributes more to the total cytosolic Ca
2+
response while influx from the extracellular medium contributes less. These results provide the first molecular genetic evidence that release of Ca
2+
from the ER contributes to cytosolic Ca
2+
responses in
D. discoideum
.
Characterization of Farnesylated Protein Tyrosine Phosphatase TcPRL-1 from
Trypanosoma cruzi
Authors: Cuevas Ileana C. , Rohloff Peter , Sánchez Daniel O. , Docampo Roberto
Published: September 2005

Protein tyrosine kinases and phosphatases play important roles in the regulation of cell growth, development, and differentiation. We report here the identification in
Trypanosoma cruzi
of a gene (
TcPRL-1
) encoding a protein tyrosine phosphatase. The predicted protein (TcPRL-1) shares ca. 35% identity with the mammalian protein tyrosine phosphatase known as phosphatase of regenerating liver 1 (PRL-1). Four copies of this protein tyrosine phosphatase are present in the
T. cruzi
genome, and Northern blot assays showed a transcript of ∼750 bases. TcPRL-1 was detected by Western blot analysis only in amastigote extracts as a 21-kDa protein. TcPRL-1 was expressed in
Escherichia coli
, and its phosphatase activity was determined by using
p
-nitrophenylphosphate and a phosphorylated protein as substrates. In contrast to other PRLs, TcPRL-1 activity was not affected by pentamidine, and it was inhibited by very low concentrations of
o
-vanadate. TcPRL-1 has a C-terminal CAAX motif (CAVM) and is farnesylated in vitro by
T. cruzi
epimastigote extracts and in vivo according to the transfection results. After transfection of
T. cruzi
with a vector that expresses TcPRL-1 as a C-terminal fusion to green fluorescent protein, GFP-TcPRL-1 was detected in the endocytic pathway of epimastigotes, amastigotes, and trypomastigotes by colocalization with cruzipain and concanavalin A. Interestingly, a mutant form without the CAAX motif localized to the cytoplasm, in contrast to its mammalian counterparts that localize to the nucleus. The results of these studies on TcPRL-1 reveal that, even though the animal and parasite PRLs share similar kinetic properties, their susceptibilities to inhibitors, as well as their localization, are distinct, implying that they may be involved in different cellular processes.
Genome-Wide Transcription Profiling of the Early Phase of Biofilm Formation by
Candida albicans
Authors: Murillo Luis A. , Newport George , Lan Chung-Yu , Habelitz Stefan , Dungan Jan , Agabian Nina M.
Published: September 2005

The ability to adhere to surfaces and develop as a multicellular community is an adaptation used by most microorganisms to survive in changing environments. Biofilm formation proceeds through distinct developmental phases and impacts not only medicine but also industry and evolution. In organisms such as the opportunistic pathogen
Candida albicans
, the ability to grow as biofilms is also an important mechanism for persistence, facilitating its growth on different tissues and a broad range of abiotic surfaces used in medical devices. The early stage of
C. albicans
biofilm is characterized by the adhesion of single cells to the substratum, followed by the formation of an intricate network of hyphae and the beginning of a dense structure. Changes in the transcriptome begin within 30 min of contact with the substrate and include expression of genes related to sulfur metabolism, in particular
MET3
, and the equivalent gene homologues of the Ribi regulon in
Saccharomyces cerevisiae
. Some of these changes are initiated early and maintained throughout the process; others are restricted to the earliest stages of biofilm formation. We identify here a potential alternative pathway for cysteine metabolism and the biofilm-associated expression of genes involved in glutathione production in
C. albicans
.
Characterization of a Molecular Chaperone Present in the Eukaryotic Flagellum
Authors: Shapiro Jessica , Ingram Jessica , Johnson Karl A.
Published: September 2005

Chlamydomonas
flagella contain a molecular chaperone now identified as HSP70A, a major cytoplasmic isoform. HSP70A synthesis is upregulated by deflagellation, and its distribution in the flagellum overlaps with the IFT kinesin-II motor FLA10. HSP70A may chaperone flagellar proteins during transport, participating in the assembly and maintenance of the flagellum.
Genetic Analysis of the TOR Pathway in
Aspergillus nidulans
Authors: Fitzgibbon Gregory J. , Morozov Igor Y. , Jones Meriel G. , Caddick Mark X.
Published: September 2005

We identified five genes encoding components of the TOR signaling pathway within
Aspergillus nidulans
. Unlike the situation in
Saccharomyces cerevisiae
, there is only a single Tor kinase, as in plant and animal systems, and mutant phenotypes suggest that the TOR pathway plays only a minor role in regulating nitrogen metabolism.
Electronic Spectra and Ionization Potentials of Halogen Oxides Using the Fock Space Coupled-Cluster Method
Authors: Vaval Nayana , Manohar Prashant , Pal Sourav
Published: September 2005

The ionization potentials and excitation energies of halogen monoxides and dihalogen oxides are studied using fully size-extensive Fock space version of multi-reference coupled-cluster theory. The low-lying excited states of ClO-, FO-, Cl2O and F2O are obtained along with the ionization spectra of Cl2O and F2O. The adiabatic electron affinity of ClO, FO and the vertical detachment energy of ClO- and FO- are also presented.
A Class of Exactly Solvable Schrödinger Equations
Authors: Karwowski Jacek , Cyrnek Lech
Published: September 2005

An algebraic approach to solving a class of one-particle Schrödinger equations is presented. As an example, quasi-exact solutions of the eigenvalue problem of a Hamiltonian describing two interacting particles confined in a parabolic well are obtained. This example constitutes a unification and a generalization of several models known in the literature, as the ones of Taut (Phys. Rev. A 1993, 48, 3561) and of Samanta and Ghosh (Phys. Rev. A 1990, 42, 1178). Two confined particles interacting by Coulomb forces and the nuclear motion of a diatomic molecule are discussed as practical implementations.
Theoretical Fine Spectroscopy with SAC-CI Method: Outer- and Inner-Valence Ionization Spectra of CO and N2
Authors: Ehara Masahiro , Ishida Mayumi , Nakatsuji Hiroshi
Published: September 2005

Outer- and inner-valence ionization spectra of CO and N2 were studied by the SAC-CI (symmetry-adapted-cluster configuration-interaction) general-R method. Fine details of experimental spectra of these molecules were reproduced and quantitative assignments of the peaks were proposed. Both outer- and inner-valence satellites were classified into the shake-up states including the valence or Rydberg excitations. For CO, theoretical satellite spectrum up to 50 eV was presented and the nine bands of 22-50 eV observed by EMS and eight bands of 22-34 eV by XPS (C-K) were characterized in detail. Numerous satellite peaks with distributed intensity were obtained and some of them, especially for bands 4, 5, 6 and 7, were predominantly described by triple-electron processes. For N2, the spectrum up to 45 eV was calculated and the complex satellite peaks observed by XPS in the lower energy region 20-33 eV were interpreted. The detailed assignments for the (2σg-1) satellite states in the higher-energy region 33-45 eV were also presented.
Borrowing Intensity in Rare Earth Doped Materials; Magnetic Dipole Transitions
Authors: Wybourne Brian G. , Smentek Lidia , Kędziorski Andrzej
Published: September 2005

A theoretical model of magnetic dipole transitions in crystals doped by the lanthanide ions is based on the fourth-order perturbation theory applied for the Hamiltonian in which two perturbing mechanisms are taken into account. Namely, the impact due to the crystal field potential and also spin-orbit interaction operator is included in the transition amplitude. The analysis is performed in the language of Racah algebra applied for the formulation of the effective operators expressed in terms of unit double tensor operators. The radial integrals of the terms contributing at the fourth order are defined within the perturbed function approach, due to which their values are evaluated for the complete radial basis sets of one-electron states of given symmetry. The numerical analysis performed for the Eu3+ ion provides the information on the relative importance of various effective operators and allows one to establish a hierarchy of important terms contributing to the transition amplitude. The results of the analysis lead also to the verification of possible importance of magnetic dipole transitions in the description of 0 ↔ 0 and 0 ↔ 1 electric dipole transitions via the so-called borrowing mechanism introduced by Wybourne.
Coupled-Cluster Study of Spectroscopic Constants of the Alkali Metal Diatomics: Ground and the Singlet Excited States of Na2, NaLi, NaK, and NaRb
Authors: Neogrády Pavel , Szalay Péter G. , Kraemer Wolfgang P. , Urban Miroslav
Published: September 2005

A newly implemented two-determinant coupled-cluster method including single and double excitation operators (TD-CCSD) is applied to calculations of spectroscopic constants of alkali metal diatomics. The equilibrium bond length, harmonic vibrational frequency, anharmonicity, the dissociation energy and excitation energies are derived from the potential curves calculated for the ground state X1Σ+u and the two singlet mono-excited states, A1Σ+u and B1Πu of Na2 as well as for the ground X1Σ+ and the singlet excited A1Σ+ and B1Π states of the heteronuclear NaLi, NaK, and NaRb molecules. Spectroscopic constants and excitation energies agree reasonably well with experiment. Our results demonstrate that the relatively simple CCSD method for the excited states represented by two-reference determinants is a viable technique. The computer time needed for an excited singlet state is practically identical to the time which a standard single-determinant-based CCSD calculation takes.
The Hodge Operator in Fermionic Fock Space
Authors: Stolarczyk Leszek Z.
Published: September 2005

The Hodge operator ("star" operator) plays an important role in the theory of differential forms, where it serves as a tool for the switching between the exterior derivative and co-derivative. In the theory of many-electron systems involving a finite-dimensional fermionic Fock space, one can define the Hodge operator as a unique (i.e., invariant with respect to linear transformations of the spin-orbital basis set) antilinear operator. The similarity transformation based on the Hodge operator results in the switching between the fermion creation and annihilation operators. The present paper gives a self-contained account on the algebraic structures which are necessary for the construction of the Hodge operator: the fermionic Fock space, the corresponding Grassmann algebra, and the generalized creation and annihilation operators. The Hodge operator is then defined, and its properties are reviewed. It is shown how the notion of the Hodge operator can be employed in a construction of the electronic time-reversal operator.
Multireference State-Specific Coupled-Cluster Theory and Multiconfigurationality Index. BH Dissociation
Authors: Ivanov Vladimir V. , Adamowicz Ludwik , Lyakh Dmitry I.
Published: September 2005

Multiconfigurationality index calculated for the coupled-cluster wave function based on an algorithm developed using a computer-aided generation approach is applied to analyze the multireference state-specific coupled-cluster method with the CAS reference (i.e. the so called the CAS(n,m)CCSD approach). The numerical results concern dissociation of the BH molecule where at larger displacement from the equilibrium significant quasi-degeneracy arises. The analysis shows that the CAS(n,m)CCSD approach performs very well in such a situation.
PREVENTIVE CARE FOR CHILDREN IN THE UNITED STATES: Quality and Barriers
Authors: Chung Paul J. , Lee Tim C. , Morrison Janina L. , Schuster Mark A.
Published: September 2005

▪ Abstract  Our objective was to examine the academic literature covering quality of childhood preventive care in the United States and to identify barriers that contribute to poor or disparate quality. We systematically reviewed articles related to childhood preventive care published from 1994 through 2003, focusing on 58 large observational studies and interventions addressing well-child visit frequency, developmental and psychosocial surveillance, disease screening, and anticipatory guidance. Although many children attend recommended well-child visits and receive comprehensive preventive care at those visits, many do not attend such visits. Estimates of children who attend all recommended visits range widely (from 37%–81%). In most studies, less than half is the proportion of children who receive developmental or psychosocial surveillance, adolescents who are asked about various health risks, children at risk for lead exposure who are screened, adolescents at risk for Chlamydia who are tested, or children and adolescents who receive anticipatory guidance on various topics. Major barriers include lack of insurance, lack of continuity with a clinician or place of care, lack of privacy for adolescents, lack of clinician awareness or skill, racial/ethnic barriers, language-related barriers, clinician and patient gender-related barriers, and lack of time. In summary, childhood preventive care quality is mixed, with large disparities among populations. Recent research has identified barriers that might be overcome through practice and policy interventions.
FOR-PROFIT CONVERSION OF BLUE CROSS PLANS: Public Benefit or Public Harm?
Authors: Hall Mark A. , Conover Christopher J.
Published: September 2005

▪ Abstract  Conversions of Blue Cross plans to for-profit status have the potential to remake the corporate landscape of health care finance. Absent regulatory intervention, current trends could easily result in more than half of Blue Cross subscribers being in for-profit plans, a phenomenon far more significant than the conversion of nonprofit hospitals. Therefore, regulators' deliberations over conversion proposals are beginning to focus on the health policy impacts. This chapter surveys the full range of health policy implications by analyzing all existing studies of Blue Cross conversions and reporting on the authors' own case studies of conversion impacts. These studies conclude that conversions have not caused major negative impacts on the availability or accessibility of health care in the states in which conversions have occurred so far. However, a great deal of uncertainty exists about the actual effects of previous conversions, and each state is unique; therefore, even if the historical record were clear, it is difficult to predict with great certainty what the actual effects will be in another state undergoing a Blue conversion.
VACCINE SHORTAGES: History, Impact, and Prospects for the Future
Authors: Hinman Alan R. , Orenstein Walter A. , Santoli Jeanne M. , Rodewald Lance E. , Cochi Stephen L.
Published: September 2005

▪ Abstract  Vaccine shortages can result from higher-than-expected demand, interruptions in production/supply, or a lack of resources to purchase vaccines. Each of these factors has played a role in vaccine shortages in the United States during the past 20 years. Since 2000, the United States has experienced an unprecedented series of shortages of vaccines recommended for widespread use against 9 diseases, after more than 15 years without vaccine supply problems. In developing countries, the major cause of vaccine shortages is lack of resources to purchase them. Although there are several steps that could reduce the likelihood of future vaccine shortages, many would take several years to implement. Consequently, we will probably continue to see occasional shortages of vaccines in the United States in the next few years.