bluuebunny/crossref_metadata_2025_split huggingface dataset:
Apaf‐1 expression in human cutaneous melanoma progression and in pigmented nevi
Authors: Niedojadło Katarzyna , Łabędzka Karolina , Łada Ewelina , Milewska Agnieszka , Chwirot Barbara W. 
Published: November 2005

SummaryMalignant melanoma is notoriously refractive to therapy and resistant to apoptosis. This may reflect the downregulation of Apaf‐1, an important mediator of mitochondrial‐dependent apoptosis, observed in vitro in melanoma cell lines and by immunohistochemistry for Apaf‐1 protein in histological samples of primary and metastatic melanomas. Although it has been suggested that loss of Apaf‐1 expression may be an indicator of malignant transformation in melanoma, previous studies on Apaf‐1 expression in benign pigmented nevi were performed without reference to their histologic type. Here we have evaluated the expression of Apaf‐1 mRNA by fluorescence in situ hybridization and of Apaf‐1 protein by immunohistochemistry in a large panel of human melanomas and in eight types of pigmented nevi, considered potential precursors for cutaneous melanoma. We observe a strong negative correlation between melanoma progression assessed according to Clark classification and the expression of Apaf‐1. A significant decrease in Apaf‐1 expression was observed between Clark II and Clark III lesions, the stages usually associated with a transition from horizontal to vertical growth phase of melanoma. There was also statistically significant difference in Apaf‐1 mRNA expression between melanomas of Breslow thickness <1 mm and >4 mm. No Apaf‐1 expression could be detected in lymph node melanoma metastases. These results suggest that Apaf‐1 expression may become a prognostic marker for progress of human cutaneous melanoma and further support the notion that loss of Apaf‐1 may be an important contributory factor in the development of the disease.
Novel 4-Chlorophenol Degradation Gene Cluster and Degradation Route via Hydroxyquinol in
Arthrobacter chlorophenolicus
A6
Authors: Nordin Karolina , Unell Maria , Jansson Janet K. 
Published: November 2005

ABSTRACT

Arthrobacter chlorophenolicus
A6, a previously described 4-chlorophenol-degrading strain, was found to degrade 4-chlorophenol via hydroxyquinol, which is a novel route for aerobic microbial degradation of this compound. In addition, 10 open reading frames exhibiting sequence similarity to genes encoding enzymes involved in chlorophenol degradation were cloned and designated part of a chlorophenol degradation gene cluster (
cph
genes). Several of the open reading frames appeared to encode enzymes with similar functions; these open reading frames included two genes,
cphA-I
and
cphA-II
, which were shown to encode functional hydroxyquinol 1,2-dioxygenases. Disruption of the
cphA-I
gene yielded a mutant that exhibited negligible growth on 4-chlorophenol, thereby linking the
cph
gene cluster to functional catabolism of 4-chlorophenol in
A. chlorophenolicus
A6. The presence of a resolvase pseudogene in the
cph
gene cluster together with analyses of the G+C content and codon bias of flanking genes suggested that horizontal gene transfer was involved in assembly of the gene cluster during evolution of the ability of the strain to grow on 4-chlorophenol.
Preparation, Characterization, and Optimization of an In Vitro C30Carotenoid Pathway
Authors: Ku Bosung , Jeong Jae-Cheol , Mijts Benjamin N. , Schmidt-Dannert Claudia , Dordick Jonathan S. 
Published: November 2005

ABSTRACTTheispAgene encoding farnesyl pyrophosphate (FPP) synthase fromEscherichia coliand thecrtMgene encoding 4,4′-diapophytoene (DAP) synthase fromStaphylococcus aureuswere overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP. This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoid-based pathways, could lead to the synthesis of novel carotenoids and their derivatives.
Genetic and Phylogeographic Structures of the Symbiotic Fungus
Tuber magnatum
Authors: Rubini Andrea , Paolocci Francesco , Riccioni Claudia , Vendramin Giovanni G. , Arcioni Sergio 
Published: November 2005

ABSTRACT

The quality and market price of truffles vary with the species and, traditionally, the place of origin. The premium species
Tuber magnatum
produces white truffles and has a patchy distribution restricted to Italy and some Balkan areas. We used polymorphic microsatellites to evaluate 316 specimens grouped into 26 populations sampled across the species' geographic range to determine if natural populations of
T. magnatum
are genetically differentiated. We found that the southernmost and the northwesternmost populations were significantly differentiated from the rest of the populations. The simple sequence repeat data also could be used to make inferences about the postglacial
T. magnatum
expansion pattern. This study is the first to identify a genetic and phylogeographic structure in
T. magnatum.
The presence of a genetic structure can be of practical interest in tracing truffle populations according to their geographic origin for marketing strategies. Evidence for extensive outcrossing in field populations of
T. magnatum
also is provided for the first time.
Intra- and Interspecific Comparisons of Bacterial Diversity and Community Structure Support Coevolution of Gut Microbiota and Termite Host
Authors: Hongoh Yuichi , Deevong Pinsurang , Inoue Tetsushi , Moriya Shigeharu , Trakulnaleamsai Savitr , Ohkuma Moriya , Vongkaluang Charunee , Noparatnaraporn Napavarn , Kudo Toshiaki 
Published: November 2005

ABSTRACT

We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four
Microcerotermes
species (Termitidae) and four
Reticulitermes
species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera
Microcerotermes
and
Reticulitermes
, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from
Microcerotermes
spp. and 367 phylotypes from
Reticulitermes
spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic “termite clusters” comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.
On-Site DNA Extraction and Real-Time PCR for Detection of
Phytophthora ramorum
in the Field
Authors: Tomlinson J. A. , Boonham N. , Hughes K. J. D. , Griffin R. L. , Barker I. 
Published: November 2005

ABSTRACT

Phytophthora ramorum
is a recently described pathogen causing oak mortality (sudden oak death) in forests in coastal areas of California and southern Oregon and dieback and leaf blight in a range of tree, shrub, and herbaceous species in the United States and Europe. Due to the threat posed by this organism, stringent quarantine regulations are in place, which restrict the movement of a number of hosts. Fast and accurate diagnostic tests are required in order to characterize the distribution of
P. ramorum
, prevent its introduction into pathogen-free areas, and minimize its spread within affected areas. However, sending samples to a laboratory for testing can cause a substantial delay between sampling and diagnosis. A rapid and simple DNA extraction method was developed for use at the point of sampling and used to extract DNAs from symptomatic foliage and stems in the field. A sensitive and specific single-round real-time PCR (TaqMan) assay for
P. ramorum
was performed using a portable real-time PCR platform (Cepheid SmartCycler II), and a cost-effective method for stabilizing PCR reagents was developed to allow their storage and transportation at room temperature. To our knowledge, this is the first description of a method for DNA extraction and molecular testing for a plant pathogen carried out entirely in the field, independent of any laboratory facilities.
Degradation of Azo Dyes by
Trametes villosa
Laccase over Long Periods of Oxidative Conditions
Authors: Zille Andrea , Górnacka Barbara , Rehorek Astrid , Cavaco-Paulo Artur 
Published: November 2005

ABSTRACT

Trametes villosa
laccase was used for direct azo dye degradation, and the reaction products that accumulated after 72 h of incubation were analyzed. Liquid chromatography-mass spectrometry (LC-MS) analysis showed the formation of phenolic compounds during the dye oxidation process as well as a large amount of polymerized products that retain azo group integrity. The amino-phenol reactions were also investigated by
13
C-nuclear magnetic resonance and LC-MS analysis, and the polymerization character of laccase was shown. This study highlights the fact that laccases polymerize the reaction products obtained during long-term batch decolorization processes with azo dyes. These polymerized products provide unacceptable color levels in effluents, limiting the application of laccases as bioremediation agents.
Detection of
Salmonella enterica
in Naturally Contaminated Liquid Eggs by Loop-Mediated Isothermal Amplification, and Characterization of
Salmonella
Isolates
Authors: Ohtsuka Kayoko , Yanagawa Keiko , Takatori Kosuke , Hara-Kudo Yukiko 
Published: November 2005

ABSTRACT

Loop-mediated isothermal amplification (LAMP) assay was effective in detecting
Salmonella enterica
in naturally contaminated liquid egg samples.
Salmonella
was detected in 110 samples taken from four egg-breaking plants. The egg samples were pre-enriched in buffered peptone water (BPW) at 37°C for 20 h. The selective enrichment was done in Rappaport-Vassiliadis or tetrathionate broth and plated onto xylose lysine deoxycholate agar and brilliant green agar, modified. In addition, the PCR assay was used to detect
Salmonella
after pre-enrichment in BPW at 37°C for 20 h. The culture method and PCR assay were compared to the LAMP assay, which was also performed after pre-enrichment in BPW. PCR failed to detect
Salmonella
in 10% of 110 samples, whereas the culture method and LAMP assay successfully identified
Salmonella
in all samples. However, the LAMP assay was found to be much more rapid than the culture method and as sensitive in detecting
Salmonella
from liquid eggs. In all of the egg-breaking plants studied,
Salmonella
was isolated on most tested days. The positive samples showed that more than 75% of the
Salmonella
strains had identical genetic patterns when analyzed by pulsed-field gel electrophoresis. This suggests that the same
Salmonella
strains having survived long periods of time in the plants were contaminating the production line. The LAMP assay is rapid, specific, and sensitive for
Salmonella
detection in liquid eggs and is able to monitor
Salmonella
contamination in egg-handling plants more reliably.
Effect of Different Carbon Sources on Community Composition of Bacterial Enrichments from Soil
Authors: Wawrik Boris , Kerkhof Lee , Kukor Jerome , Zylstra Gerben 
Published: November 2005

ABSTRACT
Soil is a highly heterogeneous matrix, which can contain thousands of different bacterial species per gram. Only a small component of this diversity (maybe <1%) is commonly captured using standard isolation techniques, although indications are that a larger proportion of the soil community is in fact culturable. Better isolation techniques yielding greater bacterial diversity would be of benefit for understanding the metabolic activity and capability of many soil microorganisms. We studied the response of soil bacterial communities to carbon source enrichment in small matrices by means of terminal restriction fragment length polymorphism (TRFLP) analysis. The community composition of replicate enrichments from soil displayed high variability, likely attributable to soil heterogeneity. An analysis of TRFLP data indicated that enrichment on structurally similar carbon sources selected for similar bacterial communities. The same analysis indicated that communities first enriched on glucose or benzoate and subsequently transferred into medium containing an alternate carbon source retained a distinct community signature induced by the carbon source used in the primary enrichment. Enrichment on leucine presented a selective challenge that was able to override the imprint left by primary enrichment on acetate. In a time series experiment community change was most rapid 18 hours after inoculation, corresponding to exponential growth. Community composition did not stabilize even 4 days after secondary enrichment. Four different soil types were enriched on four different carbon sources. TRFLP analysis indicated that in three out of four cases communities enriched on the same carbon source were more similar regardless of which soil type was used. Conversely, the garden soil samples yielded similar enrichment communities regardless of the enrichment carbon source. Our results indicate that in order to maximize the diversity of bacteria recovered from the environment, multiple enrichments should be performed using a chemically diverse set of carbon sources.
Potential for Quantifying Expression of the
Geobacteraceae
Citrate Synthase Gene To Assess the Activity of
Geobacteraceae
in the Subsurface and on Current-Harvesting Electrodes
Authors: Holmes Dawn E. , Nevin Kelly P. , O'Neil Regina A. , Ward Joy E. , Adams Lorrie A. , Woodard Trevor L. , Vrionis Helen A. , Lovley Derek R. 
Published: November 2005

ABSTRACT

The
Geobacteraceae
citrate synthase is phylogenetically distinct from those of other prokaryotes and is a key enzyme in the central metabolism of
Geobacteraceae
. Therefore, the potential for using levels of citrate synthase mRNA to estimate rates of
Geobacter
metabolism was evaluated in pure culture studies and in four different
Geobacteraceae
-dominated environments. Quantitative reverse transcription-PCR studies with mRNA extracted from cultures of
Geobacter sulfurreducens
grown in chemostats with Fe(III) as the electron acceptor or in batch with electrodes as the electron acceptor indicated that transcript levels of the citrate synthase gene,
gltA
, increased with increased rates of growth/Fe(III) reduction or current production, whereas the expression of the constitutively expressed housekeeping genes
recA
,
rpoD
, and
proC
remained relatively constant. Analysis of mRNA extracted from groundwater collected from a U(VI)-contaminated site undergoing in situ uranium bioremediation revealed a remarkable correspondence between acetate levels in the groundwater and levels of transcripts of
gltA
. The expression of
gltA
was also significantly greater in RNA extracted from groundwater beneath a highway runoff recharge pool that was exposed to calcium magnesium acetate in June, when acetate concentrations were high, than in October, when the levels had significantly decreased. It was also possible to detect
gltA
transcripts on current-harvesting anodes deployed in freshwater sediments. These results suggest that it is possible to monitor the in situ metabolic rate of
Geobacteraceae
by tracking the expression of the citrate synthase gene.
Iron Uptake Mechanisms in the Fish Pathogen
Tenacibaculum maritimum
Authors: Avendaño-Herrera Ruben , Toranzo Alicia E. , Romalde Jesús L. , Lemos Manuel L. , Magariños Beatriz 
Published: November 2005

ABSTRACT

We present here the first evidence of the presence of iron uptake mechanisms in the bacterial fish pathogen
Tenacibaculum maritimum
. Representative strains of this species, with different serotypes and origins, were examined. All of them were able to grow in the presence of the chelating agent ethylenediamine-di- (
o-
hydroxyphenyl acetic acid) (EDDHA) and also produced siderophores. Cross-feeding assays suggest that the siderophores produced are closely related. In addition, all
T. maritimum
strains utilized transferrin, hemin, hemoglobin, and ferric ammonic citrate as iron sources when added to iron-deficient media. Whole cells of all
T. maritimum
strains, grown under iron-supplemented or iron-restricted conditions, were able to bind hemin, indicating the existence of constitutive binding components located at the
T. maritimum
cell surface. This was confirmed by the observation that isolated total and outer membrane proteins from all of the strains, regardless of the iron levels of the media, were able to bind hemin, with the outer membranes showing the strongest binding. proteinase K treatment of whole cells did not affect the hemin binding, indicating that, in addition to proteins, some protease-resistant components could also bind hemin. At least three outer membrane proteins were induced in iron-limiting conditions, and all strains, regardless of their serotype, showed a similar pattern of induced proteins. The results of the present study suggest that
T. maritimum
possesses at least two different systems of iron acquisition: one involving the synthesis of siderophores and another that allows the utilization of heme groups as iron sources by direct binding.
Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Characterization of Biocatalytically Active Bacterial Strains and of Cytochrome P450 Monooxygenase Enzymes and Their Genes
Authors: Jungmann Volker , Molnár István , Hammer Philip E. , Hill D. Steven , Zirkle Ross , Buckel Thomas G. , Buckel Dagmar , Ligon James M. , Pachlatko J. Paul 
Published: November 2005

ABSTRACT

4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active
Streptomyces
strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in
Escherichia coli
and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined.
Culture-Dependent and Culture-Independent Diversity within the Obligate Marine Actinomycete Genus
Salinispora
Authors: Mincer Tracy J. , Fenical William , Jensen Paul R. 
Published: November 2005

ABSTRACT

Salinispora
is the first obligate marine genus within the order
Actinomycetales
and a productive source of biologically active secondary metabolites. Despite a worldwide, tropical or subtropical distribution in marine sediments, only two
Salinispora
species have thus far been cultivated, suggesting limited species-level diversity. To further explore
Salinispora
diversity and distributions, the phylogenetic diversity of more than 350 strains isolated from sediments collected around the Bahamas was examined, including strains cultured using new enrichment methods. A culture-independent method, using a
Salinispora
-specific seminested PCR technique, was used to detect
Salinispora
from environmental DNA and estimate diversity. Overall, the 16S rRNA gene sequence diversity of cultured strains agreed well with that detected in the environmental clone libraries. Despite extensive effort, no new species level diversity was detected, and 97% of the 105 strains examined by restriction fragment length polymorphism belonged to one phylotype (
S. arenicola
). New intraspecific diversity was detected in the libraries, including an abundant new phylotype that has yet to be cultured, and a new depth record of 1,100 m was established for the genus. PCR-introduced error, primarily from
Taq
polymerase, significantly increased clone library sequence diversity and, if not masked from the analyses, would have led to an overestimation of total diversity. An environmental DNA extraction method specific for vegetative cells provided evidence for active actinomycete growth in marine sediments while indicating that a majority of sediment samples contained predominantly
Salinispora
spores at concentrations that could not be detected in environmental clone libraries. Challenges involved with the direct sequence-based detection of spore-forming microorganisms in environmental samples are discussed.
Development of a
cob
-18S rRNA Gene Real-Time PCR Assay for Quantifying
Pfiesteria shumwayae
in the Natural Environment
Authors: Zhang Huan , Lin Senjie 
Published: November 2005

ABSTRACT

Despite the fact that the heterotrophic dinoflagellate
Pfiesteria shumwayae
is an organism of high interest due to alleged toxicity, its abundance in natural environments is poorly understood. To address this inadequacy, a real-time quantitative PCR assay based on mitochondrial cytochrome
b
(
cob
) and18S rRNA gene was developed and
P. shumwayae
abundance was investigated in several geographic locations. First,
cob
and its 5′-end region were isolated from a
P. shumwayae
culture, revealing three different copies, each consisting of an identical
cob
coding region and an unidentified region (X) of variable length and sequence. The unique sequences in
cob
and the X region were then used to develop a
P. shumwayae
-specific primer set. This primer set was used with reported
P. shumwayae
-specific 18S primers in parallel real-time PCRs to investigate
P. shumwayae
abundance from Maine to North Carolina along the U.S. east coast and along coasts in Chile, Hawaii, and China. Both genes generally gave similar results, indicating that this species was present, but at low abundance (mostly <10 cells · ml
−1
), in all the American coast locations investigated (with the exception of Long Island Sound, where which both genes gave negative results). Genetic variation was detected by use of both genes in most of the locations, and while
cob
consistently detected
P. shumwayae
or close genetic variants, some of the 18S PCR products were unrelated to
P. shumwayae
. We conclude that (i) the real-time PCR assay developed is useful for specific quantification of
P. shumwayae
, and (ii)
P. shumwayae
is distributed widely at the American coasts, but normally only as a minor component of plankton even in high-risk estuaries (Neuse River and the Chesapeake Bay).