bluuebunny/crossref_metadata_2025 huggingface dataset:
Applications of a Rapid Endospore Viability Assay for Monitoring UV Inactivation and Characterizing Arctic Ice Cores
Authors: Shafaat Hannah S. , Ponce Adrian
Published: October 2006

We have developed a rapid endospore viability assay (EVA) in which endospore germination serves as an indicator for viability and applied it to (i) monitor UV inactivation of endospores as a function of dose and (ii) determine the proportion of viable endospores in arctic ice cores (Greenland Ice Sheet Project 2 [GISP2] cores; 94 m). EVA is based on the detection of dipicolinic acid (DPA), which is released from endospores during germination. DPA concentrations were determined using the terbium ion (Tb
3+
)-DPA luminescence assay, and germination was induced by
l
-alanine addition. The concentrations of germinable endospores were determined by comparison to a standard curve. Parallel EVA and phase-contrast microscopy experiments to determine the percentage of germinable spores yielded comparable results (54.3% ± 3.8% and 48.9% ± 4.5%, respectively), while only 27.8% ± 7.6% of spores produced CFU. EVA was applied to monitor the inactivation of spore suspensions as a function of UV dose, yielding reproducible correlations between EVA and CFU inactivation data. The 90% inactivation doses were 2,773 J/m
2
, 3,947 J/m
2
, and 1,322 J/m
2
for EVA, phase-contrast microscopy, and CFU reduction, respectively. Finally, EVA was applied to quantify germinable and total endospore concentrations in two GISP2 ice cores. The first ice core contained 295 ± 19 germinable spores/ml and 369 ± 36 total spores/ml (i.e., the percentage of germinable endospores was 79.9% ± 9.3%), and the second core contained 131 ± 4 germinable spores/ml and 162 ± 17 total spores/ml (i.e., the percentage of germinable endospores was 80.9% ± 8.8%), whereas only 2 CFU/ml were detected by culturing.
Biochemical and Molecular Characterization of Tetracycline-Resistant
Aeromonas veronii
Isolates from Catfish
Authors: Nawaz Mohamed , Sung Kidon , Khan Saeed A. , Khan Ashraf A. , Steele Roger
Published: October 2006

Eighty-one tetracycline-resistant
Aeromonas
sp. strains were isolated from farm-raised catfish. Morphological and biochemical characteristics indicated that 23 of the 81 aeromonads were
Aeromonas hydrophila
, 7 isolates were
Aeromonas trota
, 6 isolates were
Aeromonas caviae
, 42 isolates were
Aeromonas veronii
, and 3 isolates were
Aeromonas jandaei
. However, the AluI and MboI restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified 1.4-kb 16S rRNA gene from all 81 tetracycline-resistant aeromonads from catfish were identical to the RFLP banding patterns of
A. veronii
ATCC 35626, indicating that all 81 isolates were strains of
A. veronii
. A multiplex PCR assay successfully amplified the 5 tetracycline-resistant genes (
tet
A to E) from the genomic DNA of all 81 isolates. The assay determined that
tet
E was the dominant gene occurring in 73/81 (90.0%) of the aeromonads. Plasmids (2.0 to 20 kb) were isolated from 33 of the 81 isolates. Dendrogram analysis of the SpeI pulsed-field gel electrophoresis identified 15 distinct macrorestriction patterns among the isolates. Our results indicate the need for use of 16S rRNA in the identification of
Aeromonas
spp. and the prevalence of catfish as a reservoir of
tet
genes.
Culture-Independent Analysis of Indomethacin-Induced Alterations in the Rat Gastrointestinal Microbiota
Authors: Dalby Andrew B. , Frank Daniel N. , St. Amand Allison L. , Bendele Alison M. , Pace Norman R.
Published: October 2006

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed for a variety of inflammatory conditions; however, the benefits of this class of drugs are accompanied by deleterious side effects, most commonly gastric irritation and ulceration. NSAID-induced ulceration is thought to be exacerbated by intestinal microbiota, but previous studies have not identified specific microbes that contribute to these adverse effects. In this study, we conducted a culture-independent analysis of ∼1,400 bacterial small-subunit rRNA genes associated with the small intestines and mesenteric lymph nodes of rats treated with the NSAID indomethacin. This is the first molecular analysis of the microbiota of the rat small intestine. A comparison of clone libraries and species-specific quantitative PCR results from rats treated with indomethacin and untreated rats revealed that organisms closely related to
Enterococcus faecalis
were heavily enriched in the small intestine and mesenteric lymph nodes of the treated rats. These data suggest that treatment of NSAID-induced ulceration may be facilitated by addressing the microbiological imbalances.
Manganese(IV) Oxide Production by
Acremonium
sp. Strain KR21-2 and Extracellular Mn(II) Oxidase Activity
Authors: Miyata Naoyuki , Tani Yukinori , Maruo Kanako , Tsuno Hiroshi , Sakata Masahiro , Iwahori Keisuke
Published: October 2006

Ascomycetes that can deposit Mn(III, IV) oxides are widespread in aquatic and soil environments, yet the mechanism(s) involved in Mn oxide deposition remains unclear. A Mn(II)-oxidizing ascomycete,
Acremonium
sp. strain KR21-2, produced a Mn oxide phase with filamentous nanostructures. X-ray absorption near-edge structure (XANES) spectroscopy showed that the Mn phase was primarily Mn(IV). We purified to homogeneity a laccase-like enzyme with Mn(II) oxidase activity from cultures of strain KR21-2. The purified enzyme oxidized Mn(II) to yield suspended Mn particles; XANES spectra indicated that Mn(II) had been converted to Mn(IV). The pH optimum for Mn(II) oxidation was 7.0, and the apparent half-saturation constant was 0.20 mM. The enzyme oxidized ABTS [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] (pH optimum, 5.5;

K
m

, 1.2 mM) and contained two copper atoms per molecule. Moreover, the N-terminal amino acid sequence (residues 3 to 25) was 61% identical with the corresponding sequence of an
Acremonium
polyphenol oxidase and 57% identical with that of a
Myrothecium
bilirubin oxidase. These results provide the first evidence that a fungal multicopper oxidase can convert Mn(II) to Mn(IV) oxide. The present study reinforces the notion of the contribution of multicopper oxidase to microbially mediated precipitation of Mn oxides and suggests that
Acremonium
sp. strain KR21-2 is a good model for understanding the oxidation of Mn in diverse ascomycetes.
Use of Optical Density Detection Times To Assess the Effect of Acetic Acid on Single-Cell Kinetics
Authors: Métris A. , George S. M. , Baranyi J.
Published: October 2006

The growth of
Listeria innocua
at different acetic acid concentrations (0 to 2,000 ppm) was monitored by optical density measurements in a Bioscreen (Labsystems, Vantaa, Finland). The generated populations came from low inocula that were obtained by serial dilution. A new method to estimate both the growth rate and the lag time of single cells from the detection times (time to reach an optical density of 0.11) was developed. It assumes that the single-cell lag times follow a gamma distribution and takes into account the randomness of the inoculation level. (The initial cell number per well was assumed to follow a Poisson distribution.) In this way, relatively small numbers of replicates are sufficient to obtain a robust estimation of the distribution of single-cell lag times. The results were validated with plate count experiments. It was found that logarithms of both the growth rates and of population lag times increased linearly with the acetic acid concentration. The logarithm of the scale parameter of the gamma distribution of the single-cell lag times also increased linearly with the acetic acid concentration irrespective of the phase of the inoculum.
Host
PGRP
Gene Expression and Bacterial Release in Endosymbiosis of the Weevil
Sitophilus zeamais
Authors: Anselme Caroline , Vallier Agnès , Balmand Séverine , Fauvarque Marie-Odile , Heddi Abdelaziz
Published: October 2006

Intracellular symbiosis (endosymbiosis) with gram-negative bacteria is common in insects, yet little is known about how the host immune system perceives the endosymbionts and controls their growth and invasion without complete bacterial clearance. In this study, we have explored the expression of a peptidoglycan recognition protein gene of the weevil
Sitophilus zeamais
(
wPGRP
); an ortholog in
Drosophila
(i.e.,
PGRP-LB
) was recently shown to downregulate the Imd pathway (A. Zaidman-Remy, M. Herve, M. Poidevin, S. Pili-Floury, M. S. Kim, D. Blanot, B. H. Oh, R. Ueda, D. Mengin-Lecreulx, and B. Lemaitre, Immunity
24:
463-473, 2006). Insect challenges with bacteria have demonstrated that
wPGRP
is induced by gram-negative bacteria and that the level of induction depends on bacterial growth. Real-time reverse transcription-PCR quantification of the
wPGRP
gene transcript performed at different points in insect development has shown a high steady-state level in the bacteria-bearing organ (the bacteriome) of larvae and a high level of
wPGRP
up-regulation in the symbiotic nymphal phase. Concomitantly, during this stage fluorescence in situ hybridization has revealed an endosymbiont release from the host bacteriocytes. Together with the previously described high induction level of endosymbiont virulence genes at the nymphal phase (C. Dale, G. R. Plague, B. Wang, H. Ochman, and N. A. Moran, Proc. Natl. Acad. Sci. USA
99:
12397-12402, 2002), these findings indicate that insect mutualistic relationships evolve through an interplay between bacterial virulence and host immune defense and that the host immunity engages the
PGRP
gene family in that interplay.
Multiparameter Assessments To Determine the Effects of Sugars and Antimicrobials on a Polymicrobial Oral Biofilm
Authors: Yang Ying , Sreenivasan Prem K. , Subramanyam Ravi , Cummins Diane
Published: October 2006

Clinical studies indicate relationships between dental plaque, a naturally formed biofilm, and oral diseases. The crucial role of nonmicrobial biofilm constituents in maintaining biofilm structure and biofilm-specific attributes, such as resistance to shear and viscoelasticity, is increasingly recognized. Concurrent analyses of the diverse nonmicrobial biofilm components for multiparameter assessments formed the focus of this investigation. Comparable numbers of
Actinomyces viscosus
,
Streptococcus sanguinis
,
Streptococcus mutans
,
Neisseria subflava
, and
Actinobacillus actinomycetemcomitans
cells were seeded into multiple wells of 96-well polystyrene plates for biofilm formation. Quantitative fluorescence and confocal laser scanning microscopy (CLSM) examined the influences of dietary sugars, incubation conditions, ingredients in oral hygiene formulations, and antibiotics on biofilm components. Biofilm extracellular polymeric substances (EPS) were examined with an optimized mixture of fluorescent lectins, with biofilm proteins, lipids, and nucleic acids detected with specific fluorescent stains. Anaerobic incubation of biofilms resulted in significantly more biofilm EPS and extractable carbohydrates than those formed under aerobic conditions (
P
< 0.05). Sucrose significantly enhanced biofilm EPS in comparison to fructose, galactose, glucose, and lactose (
P
< 0.05). CLSM demonstrated thicker biofilms under sucrose-replete conditions, along with significant increases in biofilm EPS, proteins, lipids, and nucleic acids, than under conditions of sucrose deficiency (
P
< 0.05). Agents in oral hygiene formulations (chlorhexidine, ethanol, and sodium lauryl sulfate), a mucolytic agent (
N
-acetyl-
l
-cysteine), and antibiotics with different modes of action (amoxicillin, doxycycline, erythromycin, metronidazole, and vancomycin) inhibited biofilm components (
P
< 0.05). Multiparameter analysis indicated a dose-dependent inhibition of biofilm EPS and protein by chlorhexidine and sodium lauryl sulfate, along with distinctive inhibitory patterns for subinhibitory concentrations of antibiotics. Collectively, these results highlight multiparameter assessments as a broad platform for simultaneous assessment of diverse biofilm components.
Phylogeny of Acetate-Utilizing Microorganisms in Soils along a Nutrient Gradient in the Florida Everglades
Authors: Chauhan Ashvini , Ogram Andrew
Published: October 2006

The consumption of acetate in soils taken from a nutrient gradient in the northern Florida Everglades was studied by stable isotope probing. Bacterial and archaeal 16S rRNA gene clone libraries from eutrophic and oligotrophic soil microcosms strongly suggest that a significant amount of acetate is consumed by syntrophic acetate oxidation in nutrient-enriched soil.
Virulence Factor Gene Profiles of
Escherichia coli
Isolates from Clinically Healthy Pigs
Authors: Schierack Peter , Steinrück Hartmut , Kleta Sylvia , Vahjen Wilfried
Published: October 2006

Nonpathogenic, intestinal
Escherichia coli
(commensal
E. coli
) supports the physiological intestinal balance of the host, whereas pathogenic
E. coli
with typical virulence factor gene profiles can cause severe outbreaks of diarrhea. In many reports,
E. coli
isolates from diarrheic animals were classified as putative pathogens. Here we describe a broad variety of virulence gene-positive
E. coli
isolates from swine with no clinical signs of intestinal disease. The isolation of
E. coli
from 34 pigs from the same population and the testing of 331 isolates for genes encoding heat-stable enterotoxins I and II, heat-labile enterotoxin I, Shiga toxin 2e, and F4, F5, F6, F18, and F41 fimbriae revealed that 68.6% of the isolates were positive for at least one virulence gene, with a total of 24 different virulence factor gene profiles, implying high rates of horizontal gene transfer in this
E. coli
population. Additionally, we traced the occurrence of hemolytic
E. coli
over a period of 1 year in this same pig population. Hemolytic isolates were differentiated into seven clones; only three were found to harbor virulence genes. Hemolytic
E. coli
isolates without virulence genes or with only the
fedA
gene were found to be nontypeable by slide agglutination tests with OK antisera intended for screening live cultures against common pathogenic
E. coli
serogroups. The results appear to indicate that virulence gene-carrying
E. coli
strains are a normal part of intestinal bacterial populations and that high numbers of
E. coli
cells harboring virulence genes and/or with hemolytic activity do not necessarily correlate with disease.
Denaturing Gradient Gel Electrophoresis as a Diagnostic Tool in Periodontal Microbiology
Authors: Zijnge Vincent , Welling Gjalt W. , Degener John E. , van Winkelhoff Arie Jan , Abbas Frank , Harmsen Hermie J. M.
Published: October 2006

Bacteria play an important role in the initiation and progression of periodontal diseases and are part of a biofilm, which can contain over 100 different species. The aim of the present study was to show the potential of denaturing gradient gel electrophoresis (DGGE) as a tool for the detection of clinically relevant species and to compare the results of detection by DGGE with those by PCR and culturing. Hybridization of the bands from the DGGE profiles with species-specific probes was developed to confirm the band positions in the marker obtained with reference strains. The sensitivities of DGGE compared to those of cultivation for the detection of
Actinobacillus actinomycetemcomitans
,
Porphyromonas gingivalis
,
Prevotella intermedia
, and
Tannerella forsythensis
were 100, 100, 88, and 100%, respectively; and the sensitivities of DGGE compared to those of PCR were 100, 90, 88, and 96%, respectively. DGGE as a diagnostic tool could easily be extended to other species, as shown for
Treponema denticola
, which could be detected in 48% of the samples. Three different groups of
A. actinomycetemcomitans
serotypes could be distinguished by DGGE (i.e., a group comprising serotypes a, d, e, and f; a group comprising serotype b; and a group comprising serotype c). Amplicons from
P. gingivalis
and
T. denticola
migrated to the same position in the gel, and
P. intermedia
produced multiple bands. In the present study we show that the DGGE profiles represent clinically relevant species which can be detected by hybridization with species-specific probes. With DGGE, large numbers of samples can be analyzed for different species simultaneously, and DGGE may be a good alternative in periodontal microbial diagnostics.
Contemporary Prevalence of BRO β-Lactamases in
Moraxella catarrhalis
: Report from the SENTRY Antimicrobial Surveillance Program (North America, 1997 to 2004)
Authors: Deshpande Lalitagauri M. , Sader Helio S. , Fritsche Thomas R. , Jones Ronald N.
Published: October 2006

A total of 7,860 community-acquired
Moraxella catarrhalis
isolates (SENTRY Antimicrobial Surveillance Program, 1997 to 2004) were tested by broth microdilution methods, and 399 randomly selected strains from North American sites were tested for BRO-1 and BRO-2 by PCR methods. Several antimicrobials remained very active, including amoxicillin-clavulanate (MIC
90
s, ≤0.25 μg/ml), azithromycin (MIC
90
s, ≤0.12 μg/ml), ceftriaxone (MIC
90
s, 0.5 μg/ml), and levofloxacin (MIC
90
s, ≤0.03 to 0.06 μg/ml). The BRO-2 incidence rates by year were 3 to 4% overall (96 to 97% for BRO-1) and were the highest in Canada (7.9%), with the incidence in the United States being only 2.0%.
Mycobacterium barrassiaesp. nov., aMycobacterium moriokaenseGroup Species Associated with Chronic Pneumonia
Authors: Adékambi Toïdi , Raoult Didier , Drancourt Michel
Published: October 2006

Three identical isolates of new rapidly growing mycobacteria (RGM) were recovered from the bronchial aspirate and sputum from a 49-year-old woman presenting with lung lesions. The case met the American Thoracic Society criteria for the diagnosis of nontuberculous mycobacterial infection. The three isolates grew in 3 days at 24 to 42°C. The 16S rRNA gene sequence analysis indicated that the sequences of the isolates were identical and shared 99.7% and 98.1% similarities with those ofMycobacterium moriokaenseandMycobacterium gadium, respectively. Partial 723-bprpoBsequence analyses indicated that the sequences of the isolates shared 95.8% and 92.3% similarities with those ofM. moriokaenseandM. gadium, respectively. Polyphasic identification (including biochemical tests; antimicrobial susceptibility profiling; andhsp65,recA, andsodAgene sequence analyses, as well as G+C content determination and cell wall fatty acid composition analysis) supported the evidence that these isolates were representative of a new species. Phylogenetic analyses confirmed the close relationships of the isolates withM. moriokaenseand the definedM. moriokaensegroup. These isolates were susceptible to the antimicrobials currently recommended for the treatment of RGM infections. These isolates differed fromM. moriokaenseby their susceptibility to vancomycin. We propose the nameMycobacterium barrassiaesp. nov. for this new species. The type strain is N7T(CIP 108545Tand CCUG 50398T).
Candida rugosa
, an Emerging Fungal Pathogen with Resistance to Azoles: Geographic and Temporal Trends from the ARTEMIS DISK Antifungal Surveillance Program
Authors: Pfaller M. A. , Diekema D. J. , Colombo A. L. , Kibbler C. , Ng K. P. , Gibbs D. L. , Newell V. A. ,   the Global Antifungal Surveillance Group
Published: October 2006

Candida rugosa
is a fungus that appears to be emerging as a cause of infection in some geographic regions. We utilized the extensive database of the ARTEMIS DISK Antifungal Surveillance Program to describe the geographic and temporal trends in the isolation of
C. rugosa
from clinical specimens and the in vitro susceptibilities of 452 isolates to fluconazole and voriconazole.
C. rugosa
accounted for 0.4% of 134,715 isolates of
Candida
, and the frequency of isolation increased from 0.03% to 0.4% over the 6.5-year study period (1997 to 2003).
C. rugosa
was most common in the Latin American region (2.7% versus 0.1 to 0.4%). Decreased susceptibility to fluconazole (40.5% susceptible) was observed in all geographic regions; however, isolates from Europe and North America were much more susceptible (97 to 100%) to voriconazole than those from other geographic regions (55.8 to 58.8%).
C. rugosa
was most often isolated from blood and urine in patients hospitalized at the Medical and Surgical inpatient services. Notably, bloodstream isolates were the least susceptible to both fluconazole and voriconazole.
C. rugosa
should be considered, along with the established pathogens
Candida krusei
and
Candida glabrata
, as a species of
Candida
with reduced susceptibility to the azole antifungal agents.
Treatment Failure of Nosocomial Pertussis Infection in a Very-Low-Birth-Weight Neonate
Authors: Bonacorsi Stéphane , Farnoux Caroline , Bidet Philippe , Caro Valérie , Aizenfisz Sophie , Benhayoun Mounir , Aujard Yannick , Guiso Nicole , Bingen Edouard
Published: October 2006

We describe a case of nosocomial maternal transmission of
Bordetella pertussis
to a very-low-birth-weight (VLBW) neonate in whom treatment was unsuccessful. This case underscores the need for rapid and sensitive PCR diagnosis in VLBW neonates and in parents with clinical signs of pertussis and suggests that standard treatment may not be appropriate for VLBW neonates.
Development of an Enzyme Immunoassay for Detection of Sapovirus-Specific Antibodies and Its Application in a Study of Seroprevalence in Children
Authors: Farkas Tibor , Deng Xiaoyun , Ruiz-Palacios Guillermo , Morrow Ardythe , Jiang Xi
Published: October 2006

Sapoviruses (SVs) are an important cause of acute pediatric gastroenteritis. Due to the lack of appropriate diagnostic methods, the epidemiology of SV-associated illness remains poorly understood. Baculovirus and
Escherichia coli
expression systems were evaluated for the development of antibody detection enzyme immunoassays (EIA). Age-related antibody prevalence in children was studied using the new EIA. Because of the low yield of the baculovirus system, the
E. coli
-expressed SV capsid proteins were used to develop the EIA. The antigenic specificities of the
E. coli
-expressed SV capsid proteins were demonstrated using hyperimmune antisera raised in animals and sera collected from patients. A high prevalence (>90%) of antibodies to both SV (strain Mex340) and norovirus (strain VA387) was observed in children involved in a birth cohort at 20 to 24 months of age; however, at 1 to 3 months of age, <25% of the children possessed anti-SV antibodies versus >90% with anti-NV antibodies. The
E. coli
-derived SV proteins are an excellent source of antigens for the EIA. SV infection is common in the first 2 years of life. The low prevalence of maternal antibodies detected in Mexican children against SVs in this study is unique and needs to be addressed in future studies.
Detection of Norovirus Antigens from Recombinant Virus-Like Particles and Stool Samples by a Commercial Norovirus Enzyme-Linked Immunosorbent Assay Kit
Authors: Okitsu-Negishi Shoko , Okame Michio , Shimizu Yuko , Phan Tung Gia , Tomaru Takeshi , Kamijo Shigenori , Sato Takashi , Yagyu Fumihiro , Müller Werner E. G. , Ushijima Hiroshi
Published: October 2006

The commercial norovirus enzyme-linked immunosorbent assay kit was evaluated for its reactivity to recombinant virus-like particles and the detection of natural viruses from stool samples of Japanese infants and children with sporadic acute gastroenteritis compared to reverse transcription-PCR. The kit had a sensitivity of 76.3% and a specificity of 94.9%. Our results clearly indicated that the kit allows the detection of the most prevalent genotype, GII/4. In order to increase the sensitivity of the kit, the reactivity with norovirus of GII/3 and GII/6 genotypes needs to be improved.
Simplified detection of microsatellite instability in colorectal cancer without the need for corresponding germline DNA analysis: Figure 1
Authors: Ebinger M , Sotlar K , Weber A , Bock C T , Bültmann B D , Kandolf R
Published: October 2006

A panel of five quasimonomorphic mononucleotide repeats that dispenses with the need to analyse corresponding germline DNA was proposed by Suraweeraet alfor the detection of high-frequency microsatellite instability (MSI) in colorectal cancer. Using this panel, a simplified and a more sensitive (compared with the original) algorithm (p<0.05) was developed to define the instability of each repeat by assessing the morphological shape of its plot and not its absolute length. 103 cases of colorectal tumours were investigated and the results compared with those obtained by the analysis of five consensus microsatellites (Bethesda reference panel). By the proposed method, a higher specificity, but no loss of sensitivity, was found. Thus, the use of the five mononucleotide repeats in combination with the modified assessment technique simplifies the assessment of MSI, while retaining the sensitivity of the Bethesda panel for the detection of high-frequency MSI.
A new case of Carney triad: gastrointestinal stromal tumours and leiomyoma of the oesophagus do not show activating mutations of KIT and platelet-derived growth factor receptor α
Authors: Knop S , Schupp M , Wardelmann E , Stueker D , Horger M S , Kanz L , Einsele H , Kroeber S M
Published: October 2006

The Carney triad is a rare syndrome of unknown aetiology, with synchronous or metachronous appearance of rare neoplasms: gastrointestinal stromal tumours (GISTs), pulmonary chondromas and extra-adrenal paragangliomas. In most cases, the Carney triad is incomplete. The combination encountered typically, GISTs and pulmonary chondromas, was also seen in our patient, a 22-year-old woman. She was diagnosed with the triad after Billroth II gastrectomy for histologically proved gastric GISTs. The diagnosis of pulmonary chondromas was confirmed by transthoracic, computed tomography-guided needle biopsy. An oesophageal leiomyoma was resected 2 years after the initial diagnosis, on suspicion of paraganglioma. The clinical course of the patient has been uneventful since. The last follow-up was carried out 6 years after the initial diagnosis. On histological examination, the cells of gastric GIST were partly positive for CD34, whereas CD117 was expressed in all areas in variable intensity and S-100 protein was negative. The oesophageal tumour was classified as leiomyoma due to strong immunopositivity for smooth muscle actin and desmin, being negative for CD34 and CD117. Two different gastric GIST lesions as well as the oesophageal leiomyoma and normal tissue were analysed for activating mutations in common hot spots ofKIT(exon 9 and 11) and platelet-derived growth factor receptor α (exon 18), but in all probes wild-type sequences were found. These results are in accordance with the first published analyses of GIST lesions from Carney patients.
Occupational risk factors for prostate cancer and benign prostatic hyperplasia: a case–control study in Western Australia
Authors: Fritschi L , Glass D C , Tabrizi J S , Leavy J E , Ambrosini G L
Published: October 2006

Objective:To assess the association of selected occupational exposures with risk of prostate cancer and with risk of benign prostatic hyperplasia (BPH).

Methods:This population-based case–control study recruited 606 men with a diagnosis of confirmed prostate cancer, 400 men who had undergone their first prostatectomy for BPH and 471 male controls randomly selected from the electoral roll between 1 August 2001 and 1 October 2002 in Western Australia. χ2tests and logistic regressions were used for univariate and multivariate analyses to investigate the association of the two outcomes with occupational exposure to pesticides, fertilisers, metals, wood dust, oils, diesel exhaust and polyaromatic hydrocarbons (PAHs).

Results:Exposure to toxic metals at a non-substantial level increased the risk of BPH (odds ratio (OR) 1.39, 95% confidence interval (CI) 1.1 to 1.84) and led to a non-significant excess risk of prostate cancer (OR 1.25, 95% CI 0.96 to 1.61). Non-significant excess risks were observed for prostate cancer after exposure to oils other than mineral oil (OR 1.54, 95% CI 0.95 to 2.51) and for BPH after exposure to PAHs (OR 1.20, 95% CI 0.91 to 1.58). A non-statistically significant protective effect for prostate cancer was seen after exposure to organophosphate pesticides (OR 0. 69, 95% CI 0.43 to 1.12). No other associations were found for either prostate cancer or BPH and no dose–response relationships were seen for the exposures investigated.

Conclusions:These results do not provide evidence that any of the occupational factors examined are risk factors for either prostate cancer or BPH.
Administration of poly-d-glutamic acid induces proliferation of erythropoietin-producing peritubular cells in rat kidney
Authors: Kishore Bellamkonda K. , Isaac Jorge , Westenfelder Christof
Published: October 2006

Erythropoietin (EPO), a 34-kDa glycoprotein, is produced predominantly by peritubular interstitial cells (PIC) in the renal cortex and is physiologically released when ambient oxygen pressure falls. However, the exact nature of EPO-producing cells in the kidney is not well understood. We discovered that brief administration of a low-molecular-weight synthetic peptide, poly-d-glutamic acid (PDG), induced prompt and robust expansion of EPO-producing PIC in rat kidney, without evidence of tubular cell necrosis/apoptosis or fibrotic reaction. Proliferating PIC in PDG-treated rats were noninflammatory, α-smooth muscle actin negative, and specifically expressed CD73 (ecto-5′-nucleotidase), EPO mRNA, and protein. Increased numbers of EPO-positive PIC persisted even after the cessation of PDG treatment. No erythropoietic effects of EPO were detected, potentially suggesting maintained physiological control of EPO secretion in this normoxic model. We showed previously that PDG is readily filtered and is rapidly taken up and stored in lysosomes of proximal tubular cells (PTC), resulting in an apparently nonnoxious lysosomal storage condition by virtue of its nonhydrolyzable nature (Kishore BK, Maldague P, Tulkens PM, Courtoy PJ. Lab Invest 74: 1013–1023, 1996). Based on these findings, we suggest that unknown signaling molecules, produced by PTC in response to lysosomal PDG storage, appear to specifically stimulate the proliferation of EPO-producing PIC. We conclude that this model is uniquely suited to investigate the biology of EPO production by PIC and may thus facilitate the development of novel and more economical therapies of anemias and other EPO-responsive conditions.
WNK4 enhances TRPV5-mediated calcium transport: potential role in hypercalciuria of familial hyperkalemic hypertension caused by gene mutation ofWNK4
Authors: Jiang Yi , Ferguson William B. , Peng Ji-Bin
Published: October 2006

The epithelial Ca2+channel TRPV5 serves as a gatekeeper for active Ca2+reabsorption in the distal convoluted tubule and connecting tubule of the kidney. WNK4, a protein serine/threonine kinase with gene mutations that cause familial hyperkalemic hypertension (FHH), including a subtype with hypercalciuria, is also localized in the distal tubule of the nephron. To understand the role of WNK4 in modulation of Ca2+reabsorption, we evaluated the effect of WNK4 on TRPV5-mediated Ca2+transport in Xenopus laevis oocytes. Coexpression of TRPV5 with WNK4 resulted in a twofold increase in TRPV5-mediated Ca2+uptake. The increase in Ca2+uptake was due to the increase in surface expression of TRPV5. When the thiazide-sensitive Na+-Cl−cotransporter NCC was coexpressed, the effect of WNK4 on TRPV5 was weakened by NCC in a dose-dependent manner. Although the WNK4 disease-causing mutants E562K, D564A, Q565E, and R1185C retained their ability to upregulate TRPV5, the blocking effect of NCC was further strengthened when wild-type WNK4 was replaced by the Q565E mutant, which causes FHH with hypercalciuria. We conclude that WNK4 positively regulates TRPV5-mediated Ca2+transport and that the inhibitory effect of NCC on this process may be involved in the pathogenesis of hypercalciuria of FHH caused by gene mutation in WNK4.
Membrane localization and pH-dependent transport of a newly cloned organic cation transporter (PMAT) in kidney cells
Authors: Xia Li , Engel Karen , Zhou Mingyan , Wang Joanne
Published: October 2006

Plasma membrane monoamine transporter (PMAT) is a novel membrane transporter recently cloned and characterized in our laboratory. We previously demonstrated that PMAT functions as a polyspecific organic cation transporter and efficiently transports many organic cations such as monoamine neurotransmitters and 1-methyl-4-phenylpyridinium (MPP+). In this study, we explored the role of PMAT in the renal handling of organic cations. Using a polyclonal antibody generated toward the NH2-terminal 66 amino acid residues of human PMAT, we showed that the PMAT protein (∼55 kDa) is expressed in the human kidney and is primarily targeted to the apical membranes when expressed in polarized Madin-Darby canine kidney (MDCK) cells. Using MDCK cells stably expressing human PMAT, we showed that PMAT-mediated MPP+uptake is strongly dependent on extracellular pH. Lowering extracellular pH from 7.4 to 6.6 greatly stimulated PMAT-mediated MPP+uptake, whereas elevating extracellular pH to 8.2 abolished transporter activity. Kinetic analysis revealed that the apparent Vmaxat pH 6.6 is about fourfold higher than that at pH 7.4, whereas the apparent Kmvalues were not statistically different at these two conditions. Under acidic conditions (pH 6.6), the proton ionophore, carbonyl cyanide p-trifluormethoxyphenylhydrazone, drastically reduced PMAT-mediated MPP+uptake, suggesting that the stimulatory effect of proton may be due to transporter coupling with a proton gradient. Taken together, our data suggest that PMAT is expressed on the apical membranes of renal epithelial cells and may use luminal proton gradient to drive organic cation reabsorption in the kidney.
Large-scale transcriptional analysis of bovine embryo biopsies in relation to pregnancy success after transfer to recipients
Authors: El-Sayed Ashraf , Hoelker Michael , Rings Franca , Salilew Dessie , Jennen Danyel , Tholen Ernst , Sirard Marc-André , Schellander Karl , Tesfaye Dawit
Published: October 2006

The purpose of this work is to address the relationship between transcriptional profile of embryos and the pregnancy success based on gene expression analysis of blastocyst biopsies taken prior to transfer to recipients. Biopsies (30–40% of the intact embryo) were taken from in vitro-produced day 7 blastocysts ( n = 118), and 60–70% were transferred to recipients after reexpansion. Based on the success of pregnancy, biopsies were pooled in three groups (each 10 biopsies) namely: those resulting in no pregnancy (G1), resorbed embryos (G2), and those resulting in calf delivery (G3). Gene expression analysis of these groups was performed using home-made bovine preimplantation-specific cDNA array (219 clones) and BlueChip (with ∼2,000 clones). Microarray data analysis results revealed a total of 52 and 58 genes were differentially regulated during comparison between G1 vs. G3 and G2 vs. G3. Biopsies resulted in calf delivery were enriched with genes necessary for implantation (COX2 and CDX2), carbohydrate metabolism (ALOX15), growth factor (BMP15), signal transduction (PLAU), and placenta-specific 8 (PLAC8). Biopsies from embryos resulting in resorption are enriched with transcripts involved protein phosphorylation (KRT8), plasma membrane (OCLN), and glucose metabolism (PGK1 and AKR1B1). Biopsies from embryos resulting in no pregnancy are enriched with transcripts involved inflammatory cytokines (TNF), protein amino acid binding (EEF1A1), transcription factors (MSX1, PTTG1), glucose metabolism (PGK1, AKR1B1), and CD9, which is an inhibitor of implantation. In conclusion, we generated direct candidates of blastocyst-specific genes which may play an important role in determining the fate of the embryo after transfer.
Phase I Study of Bryostatin 1 and Fludarabine in Patients with Chronic Lymphocytic Leukemia and Indolent (Non-Hodgkin's) Lymphoma
Authors: Roberts John D. , Smith Mitchell R. , Feldman Eric J. , Cragg Louise , Millenson Michael M. , Roboz Gail J. , Honeycutt Connie , Thune Rose , Padavic-Shaller Kristin , Carter W. Hans , Ramakrishnan Viswanathan , Murgo Anthony J. , Grant Steven
Published: October 2006

Purpose: Preclinical studies suggested that bryostatin 1 might potentiate the therapeutic effects of fludarabine in the treatment of hematologic malignancies. We undertook a phase I study to identify appropriate schedules and doses of bryostatin 1 and fludarabine to be used in phase II studies.

Experimental Design: Patients with chronic lymphocytic leukemia (CLL) or indolent lymphoma received fludarabine daily for 5 days and a single dose of bryostatin 1 via a 24-hour continuous infusion either before or after the fludarabine course. Doses were escalated in successive patients until recommended phase II doses for each sequence were identified on the basis of dose-limiting toxic events.

Results: Bryostatin 1 can be administered safely and tolerably with full dose fludarabine (25 mg/m2/d × 5). The recommended bryostatin 1 phase II dose is 50 μg/m2 for both sequences, bryostatin 1 → fludarabine and fludarabine → bryostatin 1. The combination is active against both CLL and indolent lymphomas with responses seen in patients who had been previously treated with fludarabine. Correlative studies do not support the hypothesis that bryostatin 1 potentiates fludarabine activity through down-regulation of protein kinase C in target cells.

Conclusions: Bryostatin 1 can be administered with full dose fludarabine, and the combination is moderately active in patients with persistent disease following prior treatment. In view of the activity of monoclonal antibodies such as the anti-CD20 monoclonal antibody rituximab in the treatment of CLL and indolent lymphomas, the concept of combining bryostatin 1 and fludarabine with rituximab warrants future consideration.
Gene Expression Profiling in Cervical Cancer: An Exploration of Intratumor Heterogeneity
Authors: Bachtiary Barbara , Boutros Paul C. , Pintilie Melania , Shi Willa , Bastianutto Carlo , Li Jian-Hua , Schwock Joerg , Zhang Wendy , Penn Linda Z. , Jurisica Igor , Fyles Anthony , Liu Fei-Fei
Published: October 2006

Purpose: To explore intratumor heterogeneity in gene expression profiles from patients with cervical cancer.

Experimental Design: A total of 33 biopsies were obtained from 11 patients, sampling between two and five different areas for each tumor. The extracted RNA was hybridized onto the Affymetrix U133 Plus 2.0 oligonucleotide chip. The variance of expression within a patient (W), between patients (B) and the total variance (T = W + B) were calculated for each ProbeSet, and the ratio W/T was used as a measure of intratumor heterogeneity. Gene Ontology functional analysis was done to assess the function of genes that had high W/T (top 10%) and low W/T (bottom 10%) values.

Results: In total, 448 ProbeSets (2.2% of the total) had W/T < 0.10, indicating low intratumor heterogeneity, and 537 ProbeSets (2.7% of the total) had W/T > 0.90, indicating high intratumor heterogeneity. In total 14,473 ProbeSets (72.4%) had higher intertumor than intratumor heterogeneity (W/T < 0.5). Genes with low intratumor heterogeneity were characterized by a statistically significant enrichment of immune-related functions (P < 0.0001). Genes with high intratumor heterogeneity were characterized by a significant tendency towards nuclear localization and nucleic acid binding (both P < 0.0001). For genes with W/T > 0.5, more than six biopsies would be required to minimize the intratumoral heterogeneity to <0.15; if W/T is 0.3 to 0.4, four biopsies are required; and for low W/T of 0.16 to 0.3, only two to three biopsies would be needed.

Conclusion: Although the intratumor heterogeneity was low for the majority of the tested ProbeSets, for many genes, multiple biopsies are required to obtain a reliable estimate of gene expression.
Gene Expression Profiles of Primary Breast Carcinomas from Patients at High Risk for Local Recurrence after Breast-Conserving Therapy
Authors: Kreike Bas , Halfwerk Hans , Kristel Petra , Glas Annuska , Peterse Hans , Bartelink Harry , van de Vijver Marc J.
Published: October 2006

Purpose: Several risk factors for local recurrence of breast cancer after breast-conserving therapy (BCT) have been identified. The identification of additional risk factors would be very useful in guiding optimal therapy and also in improving understanding of the mechanisms underlying local recurrence. We used cDNA microarray analysis to identify gene expression profiles associated with local recurrence.

Experimental Design: Using 18K cDNA microarrays, gene expression profiles were obtained from 50 patients who underwent BCT. Of these 50 patients, 19 developed a local recurrence; the remaining 31 patients were selected as controls as they were free of local recurrence at least 11 years after treatment. For 9 of 19 patients, the local recurrence was also available for gene expression profiling. Unsupervised and supervised methods of classification were used to separate patients in groups corresponding to disease outcome and to study the overall gene expression pattern of primary tumors and their recurrences.

Results: Hierarchical clustering of patients did not show any grouping reflecting local recurrence status. Supervised analysis revealed no significant set of genes that was able to distinguish recurring tumors from nonrecurring tumors. Paired-data analysis of primary tumors and local recurrences showed a remarkable similarity in gene expression profile between primary tumors and their recurrences.

Conclusions: No significant differences in gene expression between primary breast cancer tumors in patients with or without local recurrence after BCT were identified. Furthermore, analyses of primary tumors and local recurrences show a preservation of the overall gene expression pattern in the local recurrence, even after radiotherapy.
Vitamin D Inhibits the Formation of Prostatic Intraepithelial Neoplasia inNkx3.1; PtenMutant Mice
Authors: Banach-Petrosky Whitney , Ouyang Xuesong , Gao Hui , Nader Kamyar , Ji Yan , Suh Nanjoo , DiPaola Robert S. , Abate-Shen Cory
Published: October 2006

Purpose: Epidemiologic studies have shown that reduced levels of vitamin D represent a major risk factor for prostate cancer. In this report, we have examined the efficacy of 1α,25-dihydroxyvitamin D3 (1,25 D3) as a chemopreventive agent using Nkx3.1; Pten mutant mice, which recapitulate stages of prostate carcinogenesis from prostate intraepithelial neoplasia (PIN) to adenocarcinoma.

Experimental Design: 1,25 D3 (or vehicle) was delivered continuously to Nkx3.1; Pten mutant or control mice for a 4-month period beginning before (precancerous cohort) or after (cancerous cohort) these mice developed PIN. At the conclusion of the study, the mice were analyzed for the occurrence of PIN and/or cancer phenotypes by histologic analyses and immunostaining using known markers of cancer progression in these mice.

Results: We found that sustained delivery of 1,25 D3 to the Nkx3.1; Pten mutant mice resulted in a significant reduction in the formation of PIN while having no apparent effect on the control mice. Furthermore, 1,25 D3 was maximally effective when delivered before, rather than subsequent to, the initial occurrence of PIN. We further show that this 1,25 D3–mediated inhibition of PIN was coincident with up-regulation of vitamin D receptor expression in the prostatic epithelium of the mutant mice, as well as in CASP prostate epithelial cell lines developed from these mice, while having no effect on androgen receptor expression or androgen receptor signaling.

Conclusion: Our findings show the value of chemoprevention studies using Nkx3.1; Pten mutant mice, particularly for evaluating the efficacy and underlying mechanisms of potential agents and to gain insights about the optimal timing of their delivery. In particular, our study predicts that vitamin D may have differential effects during early-stage versus late-stage disease and that it is more likely to be beneficial if delivered either before the overt manifestation of clinically detectable disease or during the earliest disease stages, rather than in advanced disease. Thus, our findings support the assessment of vitamin D analogues for chemoprevention in clinical trials targeting patients with early-stage disease and also establish molecular markers that can be used in such trials to determine biological activity and to optimize further clinical trials.
A Mass Balance and Disposition Study of the DNA Methyltransferase Inhibitor Zebularine (NSC 309132) and Three of Its Metabolites in Mice
Authors: Beumer Jan H. , Joseph Erin , Egorin Merrill J. , Parker Robert S. , D'argenio David Z. , Covey Joseph M. , Eiseman Julie L.
Published: October 2006

Purpose: To elucidate the in vivo metabolic fate of zebularine (NSC 309132), a DNA methyltransferase inhibitor proposed for clinical evaluation in the treatment of cancer.

Experimental Design: Male, CD2F1 mice were dosed i.v. with 100 mg/kg 2-[14C]zebularine. At specified times between 5 and 1,440 minutes, mice were euthanized. Plasma, organs, carcass, urine, and feces were collected and assayed for total radioactivity. Plasma and urine were also analyzed for zebularine and its metabolites with a previously validated high-pressure liquid chromatography assay. A similar experiment was done with 2-[14C]uridine, the proposed primary metabolite of zebularine.

Results: Maximum plasma concentrations were 462, 306, 33.6, 21.7, and 11.5 μmol/L for total radioactivity, zebularine, uridine, uracil (each at 5 minutes), and dihydrouracil (at 15 minutes), respectively. Total radioactivity, zebularine, uridine, uracil, and dihydrouracil were rapidly eliminated from plasma, and after 45 minutes, none of the individual compounds could be quantitated by high-pressure liquid chromatography. Plasma data were consistent with sequential conversion of zebularine to uridine, uracil, and dihydrouracil. 2-Pyrimidinone was not observed. Prolonged retention of radioactivity, at concentrations higher than in plasma, was observed in tissues. Recovery of given radioactivity in urine (30.3% of dose), feces (0.4% of dose), cage wash (7.9% of dose), and tissues and carcass (6.1% of dose) after 24 hours implied that up to 55% of radioactivity was expired as 14CO2. Comparison of zebularine and uridine pharmacokinetic data indicated that ∼40% of the zebularine dose was converted to uridine.

Conclusions: Zebularine is extensively and rapidly metabolized into endogenous compounds that are unlikely to have effects at the concentrations observed.
Representing Sheared Convective Boundary Layer by Zeroth- and First-Order-Jump Mixed-Layer Models: Large-Eddy Simulation Verification
Authors: Pino David , Vilà-Guerau de Arellano Jordi , Kim Si-Wan
Published: October 2006

Dry convective boundary layers characterized by a significant wind shear on the surface and at the inversion are studied by means of the mixed-layer theory. Two different representations of the entrainment zone, each of which has a different closure of the entrainment heat flux, are considered. The simpler of the two is based on a sharp discontinuity at the inversion (zeroth-order jump), whereas the second one prescribes a finite depth of the inversion zone (first-order jump). Large-eddy simulation data are used to provide the initial conditions for the mixed-layer models, and to verify their results. Two different atmospheric boundary layers with different stratification in the free atmosphere are analyzed. It is shown that, despite the simplicity of the zeroth-order-jump model, it provides similar results to the first-order-jump model and can reproduce the evolution of the mixed-layer variables obtained by the large-eddy simulations in sheared convective boundary layers. The mixed-layer model with both closures compares better with the large-eddy simulation results in the atmospheric boundary layer characterized by a moderate wind shear and a weak temperature inversion. These results can be used to represent the flux of momentum, heat, and other scalars at the entrainment zone in general circulation or chemistry transport models.