bluuebunny/crossref_metadata_2025 huggingface dataset:
Rainfall-Induced Release of Fecal Coliforms and Other Manure Constituents: Comparison and Modeling
Authors: Guber A. K. , Shelton D. R. , Pachepsky Y. A. , Sadeghi A. M. , Sikora L. J.
Published: December 2006

Modeling release of fecal coliforms is an important component of fate and transport simulations related to environmental water quality. Manure constituents other than fecal coliforms may serve as natural tracers of fecal contamination provided that their release from manure to runoff is similar to the fecal coliform release. The objectives of this work were to compare release of fecal coliforms (FC), chloride (Cl
−
), organic carbon (OC), and water-soluble phosphorus (P) from dissolving manure and to assess the performance of three models in describing the observed release. Bovine manure was applied on 0.5- by 0.3-m bare and vegetated subplots with 20% slope on sandy loam and clay loam soils. Concentrations of Cl
−
, FC, OC, and P were measured in runoff collected from troughs at the edges of the subplots at 5-min intervals during 1-h rainfall simulations. The one-parametric exponential model and two-parametric Vadas-Kleinman-Sharpley model and Bradford-Schijven model were fitted to the data. The Bradford-Schijven model had uncorrelated parameters, one of which was linearly related to the irrigation rate, and another parameter reflected the presence or the absence of vegetation. Kinetics of the FC release from manure was similar to the release kinetics of P and OC. The Bradford-Schijven model is recommended to simulate the release of manure constituents.
Engineered Cyanophycin Synthetase (CphA) from
Nostoc ellipsosporum
Confers Enhanced CphA Activity and Cyanophycin Accumulation to
Escherichia coli
Authors: Hai Tran , Frey Kay M. , Steinbüchel Alexander
Published: December 2006

The cyanophycin (CGP) synthetase gene (
cphA
NE1
) of the transposon-induced
argL
mutant NE1 of the cyanobacterium
Nostoc ellipsosporum
, which exhibits a CGP-leaky phenotype during diazotrophical growth, was cloned and expressed in
Escherichia coli
strain TOP10. Its amino acid sequence exhibited high similarities to CphAs of other cyanobacteria. Recombinant cells of
E. coli
, which harbored a fragment comprising the complete
cphA
NE1
gene plus 400 bp of its downstream region in colinear orientation to the
lacZ
promoter, accumulated CGP up to 17 and 8.5% (wt/wt) of cellular dry matter (CDM) if cultivated in complex medium in the presence or absence of isopropyl-β-
d
-thiogalactopyranoside, respectively. Two truncated CphAs, lacking 31 (CphA
NE1del96
) or 59 (CphA
NE1del180
) amino acids of the C-terminal region, were derived from
cphA
NE1
by deleting 96 or 180 bp from its 3′ region through the introduction of stop codons. In comparison to the wild-type gene,
cphA
NE1del96
conferred about 2.1- to 2.2-fold-higher enzyme activity (up to 5.75 U/mg protein) on
E. coli
. Furthermore, these cells accumulated about twofold more CGP (up to 34.5% [wt/wt] of CDM) than cells expressing the wild-type gene. An engineered CphA possessing significantly enhanced activity and conferring the highest CGP content on
E. coli
is demonstrated. In contrast, CphA
NE1del180
was inactive and did not confer CGP accumulation on
E. coli
. Interestingly, a short conserved stretch of 4 to 5 hydrophobic amino acids is located in the protein region present in CphA
NE1del96
but absent in CphA
NE1del180
. In addition, CphA
NE1
and CphA
NE1del96
are, besides CphA from
Acinetobacter baylyi
, the only CphAs exhibiting rigid substrate specificities that do not enable the incorporation of lysine instead of arginine into CGP.
Degradation of Methanethiol by Methylotrophic Methanogenic Archaea in a Lab-Scale Upflow Anaerobic Sludge Blanket Reactor
Authors: de Bok F. A. M. , van Leerdam R. C. , Lomans B. P. , Smidt H. , Lens P. N. L. , Janssen A. J. H. , Stams A. J. M.
Published: December 2006

In a lab-scale upflow anaerobic sludge blanket reactor inoculated with granular sludge from a full-scale wastewater treatment plant treating paper mill wastewater, methanethiol (MT) was degraded at 30°C to H
2
S, CO
2
, and CH
4
. At a hydraulic retention time of 9 h, a maximum influent concentration of 6 mM MT was applied, corresponding to a volumetric loading rate of 16.5 mmol liter
−1
day
−1
. The archaeal community within the reactor was characterized by anaerobic culturing and denaturing gradient gel electrophoresis analysis, cloning, and sequencing of 16S rRNA genes and quantitative PCR. Initially, MT-fermenting methanogenic archaea related to members of the genus
Methanolobus
were enriched in the reactor. Later, they were outcompeted by
Methanomethylovorans hollandica
, which was detected in aggregates but not inside the granules that originated from the inoculum, the microbial composition of which remained fairly unchanged. Possibly other species within the
Methanosarcinacaea
also contributed to the fermentation of MT, but they were not enriched by serial dilution in liquid media. The archaeal community within the granules, which was dominated by
Methanobacterium beijingense
, did not change substantially during the reactor operation. Some of the species related to
Methanomethylovorans hollandica
were enriched by serial dilutions, but their growth rates were very low. Interestingly, the enrichments could be sustained only in the presence of MT and did not utilize any of the other typical substrates for methylotrophic methanogens, such as methanol, methyl amine, or dimethylsulfide.
Escherichia coli
Biofilms Formed under Low-Shear Modeled Microgravity in a Ground-Based System
Authors: Lynch S. V. , Mukundakrishnan K. , Benoit M. R. , Ayyaswamy P. S. , Matin A.
Published: December 2006

Bacterial biofilms cause chronic diseases that are difficult to control. Since biofilm formation in space is well documented and planktonic cells become more resistant and virulent under modeled microgravity, it is important to determine the effect of this gravity condition on biofilms. Inclusion of glass microcarrier beads of appropriate dimensions and density with medium and inoculum, in vessels specially designed to permit ground-based investigations into aspects of low-shear modeled microgravity (LSMMG), facilitated these studies. Mathematical modeling of microcarrier behavior based on experimental conditions demonstrated that they satisfied the criteria for LSMMG conditions. Experimental observations confirmed that the microcarrier trajectory in the LSMMG vessel concurred with the predicted model. At 24 h, the LSMMG
Escherichia coli
biofilms were thicker than their normal-gravity counterparts and exhibited increased resistance to the general stressors salt and ethanol and to two antibiotics (penicillin and chloramphenicol). Biofilms of a mutant of
E. coli
, deficient in σ
s
, were impaired in developing LSMMG-conferred resistance to the general stressors but not to the antibiotics, indicating two separate pathways of LSMMG-conferred resistance.
Bioinformatic, Genetic, and Biochemical Evidence that Some Glycoside Hydrolase Family 42 β-Galactosidases Are Arabinogalactan Type I Oligomer Hydrolases
Authors: Shipkowski Stephanie , Brenchley Jean E.
Published: December 2006

Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the β-galactosidases are members of four families: 1, 2, 35, and 42. Most genes encoding GHF 42 enzymes are from prokaryotes unlikely to encounter lactose, suggesting a different substrate for these enzymes. In search of this substrate, we analyzed genes neighboring GHF 42 genes in databases and detected an arrangement implying that these enzymes might hydrolyze oligosaccharides released by GHF 53 enzymes from arabinogalactan type I, a pectic plant polysaccharide. Because
Bacillus subtilis
has adjacent GHF 42 and GHF 53 genes, we used it to test the hypothesis that a GHF 42 enzyme (LacA) could act on the oligosaccharides released by a GHF 53 enzyme (GalA) from galactan. We cloned these genes, plus a second GHF 42 gene from
B. subtilis
,
yesZ
, into
Escherichia coli
and demonstrated that cells expressing LacA with GalA gained the ability to use galactan as a carbon source. We constructed
B. subtilis
mutants and showed that the increased β-galactosidase activity generated in response to the addition of galactan was eliminated by inactivating
lacA
or
galA
but unaffected by the inactivation of
yesZ
. As further demonstration, we overexpressed the LacA and GalA proteins in
E. coli
and demonstrated that these enzymes degrade galactan in vitro as assayed by thin-layer chromatography. Our work provides the first in vivo evidence for a function of some GHF 42 β-galactosidases. Similar functions for other β-galactosidases in both GHFs 2 and 42 are suggested by genomic data.
Transformation of the Green Alga
Haematococcus pluvialis
with a Phytoene Desaturase for Accelerated Astaxanthin Biosynthesis
Authors: Steinbrenner Jens , Sandmann Gerhard
Published: December 2006

Astaxanthin is a high-value carotenoid which is used as a pigmentation source in fish aquaculture. Additionally, a beneficial role of astaxanthin as a food supplement for humans has been suggested. The unicellular alga
Haematococcus pluvialis
is a suitable biological source for astaxanthin production. In the context of the strong biotechnological relevance of
H. pluvialis
, we developed a genetic transformation protocol for metabolic engineering of this green alga. First, the gene coding for the carotenoid biosynthesis enzyme phytoene desaturase was isolated from
H. pluvialis
and modified by site-directed mutagenesis, changing the leucine codon at position 504 to an arginine codon. In an in vitro assay, the modified phytoene desaturase was still active in conversion of phytoene to ζ-carotene and exhibited 43-fold-higher resistance to the bleaching herbicide norflurazon. Upon biolistic transformation using the modified phytoene desaturase gene as a reporter and selection with norflurazon, integration into the nuclear genome of
H. pluvialis
and phytoene desaturase gene and protein expression were demonstrated by Southern, Northern, and Western blotting, respectively, in 11 transformants. Some of the transformants had a higher carotenoid content in the green state, which correlated with increased nonphotochemical quenching. This measurement of chlorophyll fluorescence can be used as a screening procedure for stable transformants. Stress induction of astaxanthin biosynthesis by high light showed that there was accelerated accumulation of astaxanthin in one of the transformants compared to the accumulation in the wild type. Our results strongly indicate that the modified phytoene desaturase gene is a useful tool for genetic engineering of carotenoid biosynthesis in
H. pluvialis
.
Identification of Pyruvate Carboxylase Genes in
Pseudomonas aeruginosa
PAO1 and Development of a
P. aeruginosa
-Based Overexpression System for α
4
- and α
4
β
4
-Type Pyruvate Carboxylases
Authors: Lai Huafang , Kraszewski Jessica L. , Purwantini Endang , Mukhopadhyay Biswarup
Published: December 2006

Pyruvate carboxylase (PYC) is an ecologically, medically, and industrially important enzyme. It is widespread in all three domains of life, the archaea, bacteria, and eukarya. PYC catalyzes ATP-dependent carboxylation of pyruvate to oxaloacetate. Detailed structure-function studies of this enzyme have been hampered due to the unavailability of a facile recombinant overexpression system. Except for the α
4
enzyme from a thermophilic
Bacillus
species,
Escherichia coli
has been unsuitable for overexpression of PYCs. We show that a
Pseudomonas aeruginosa
strain carrying the T7 polymerase gene can serve as a host for the overexpression of
Mycobacterium smegmatis
α
4
PYC and
Pseudomonas aeruginosa
α
4
β
4
PYC under the control of the T7 promoter from a broad-host-range conjugative plasmid. Overexpression occurred both in aerobic (LB medium) and nitrate-respiring anaerobic (LB medium plus glucose and nitrate) cultures. The latter system presented a simpler option because it involved room temperature cultures in stationary screw-cap bottles. We also developed a
P. aeruginosa Δpyc
strain that allowed the expression of recombinant PYCs in the absence of the native enzyme. Since
P. aeruginosa
can be transformed genetically and lysed for cell extract preparation rather easily, our system will facilitate site-directed mutagenesis, kinetics, X-ray crystallographic, and nuclear magnetic resonance-based structure-function analysis of PYCs. During this work we also determined that, contrary to a previous report (C. K. Stover et al., Nature 406:959-964, 2000), the open reading frame (ORF) PA1400 does not encode a PYC in
P. aeruginosa
. The α
4
β
4
PYC of this organism was encoded by the ORFs PA5436 and PA5435.
Longitudinal Analysis of Tick Densities and
Borrelia
,
Anaplasma
, and
Ehrlichia
Infections of
Ixodes ricinus
Ticks in Different Habitat Areas in The Netherlands
Authors: Wielinga Peter R. , Gaasenbeek Cor , Fonville Manoj , de Boer Albert , de Vries Ankje , Dimmers Wim , Akkerhuis Op Jagers Gerard , Schouls Leo M. , Borgsteede Fred , van der Giessen Joke W. B.
Published: December 2006

From 2000 to 2004, ticks were collected by dragging a blanket in four habitat areas in The Netherlands: dunes, heather, forest, and a city park. Tick densities were calculated, and infection with
Borrelia burgdorferi
and
Anaplasma
and
Ehrlichia
species was investigated by reverse line blot analysis. The lowest tick density was observed in the heather area (1 to 8/100 m
2
). In the oak forest and city park, the tick densities ranged from 26 to 45/100 m
2
. The highest tick density was found in the dune area (139 to 551/100 m
2
). The infection rates varied significantly for the four study areas and years, ranging from 0.8 to 11. 5% for
Borrelia
spp. and 1 to 16% for
Ehrlichia
or
Anaplasma
(
Ehrlichia/Anaplasma
) spp.
Borrelia
infection rates were highest in the dunes, followed by the forest, the city park, and heather area. In contrast,
Ehrlichia/Anaplasma
was found most often in the forest and less often in the city park. The following
Borrelia
species were found:
Borrelia
sensu lato strains not identified to the species level (2.5%),
B. afzelii
(2.5%),
B. valaisiana
(0.9%),
B. burgdorferi
sensu stricto (0.13%), and
B. garinii
(0.13%). For
Ehrlichia/Anaplasma
species,
Ehrlichia
and
Anaplasma
spp. not identified to the species level (2.5%),
Anaplasma schotti
variant (3.5%),
Anaplasma phagocytophilum
variant (0.3%), and
Ehrlichia canis
(0.19%) were found.
E. canis
is reported for the first time in ticks in The Netherlands in this study.
Borrelia lusitaniae
,
Ehrlichia chaffeensis
, and the human granylocytic anaplasmosis agent were not detected. About 1.6% of the ticks were infected with both
Borrelia
and
Ehrlichia/Anaplasma
, which was higher than the frequency predicted from the individual infection rates, suggesting hosts with multiple infections or a possible selective advantage of coinfection.
A Real-Time PCR Method for Quantifying Viable
Ascaris
Eggs Using the First Internally Transcribed Spacer Region of Ribosomal DNA
Authors: Pecson Brian M. , Barrios José Antonio , Johnson David R. , Nelson Kara L.
Published: December 2006

Worldwide, 1.4 billion people are infected with the intestinal worm
Ascaris lumbricoides
. As a result,
Ascaris
eggs are commonly found in wastewater and sludges. The current microscopy method for detecting viable
Ascaris
eggs is time- and labor-intensive. The goal of this study was to develop a real-time quantitative PCR (qPCR) method to determine the levels of total and viable
Ascaris
eggs in laboratory solutions using the first internally transcribed spacer (ITS-1) region of ribosomal DNA (rDNA) and rRNA. ITS-1 rDNA levels were proportional to
Ascaris
egg cell numbers, increasing as eggs developed from single cells to mature larvae and ultimately reaching a constant level per egg. Treatments causing >99% inactivation (high heat, moderate heat, ammonia, and UV) eliminated this increase in ITS-1 rDNA levels and caused decreases that were dependent on the treatment type. By taking advantage of this difference in ITS-1 rDNA level between viable, larvated eggs and inactivated, single-celled eggs, qPCR results were used to develop inactivation profiles for the different treatments. No statistical difference from the standard microscopy method was found in 75% of the samples (12 of 16). ITS-1 rRNA was detected only in samples containing viable eggs, but the levels were more variable than rDNA levels and ITS-1 rRNA could not be used for quantification. The detection limit of the rDNA-based method was approximately one larvated egg or 90 single-celled eggs; the detection limit for the rRNA-based method was several orders of magnitude higher. The rDNA qPCR method is promising for both research and regulatory applications.
Hypervirulence of a Rough Variant of the
Mycobacterium abscessus
Type Strain
Authors: Catherinot E. , Clarissou J. , Etienne G. , Ripoll F. , Emile J.-F. , Daffé M. , Perronne C. , Soudais C. , Gaillard J.-L. , Rottman M.
Published: December 2006

We isolated a rough variant of
Mycobacterium abscessus
CIP 104536T during experimental infection of mice. We show that this variant has lost the ability to produce glycopeptidolipids, is hyperlethal for C57BL/6 mice infected intravenously, and induces a strong tumor necrosis factor-alpha response by murine monocyte-derived macrophages.
Protective Effect of the Nramp1 BB Genotype againstBrucella abortusin the Water Buffalo (Bubalus bubalis)
Authors: Capparelli Rosanna , Alfano Flora , Amoroso Maria Grazia , Borriello Giorgia , Fenizia Domenico , Bianco Antonio , Roperto Sante , Roperto Franco , Iannelli Domenico
Published: December 2006

We tested 413 water buffalo cows (142 cases and 271 controls) for the presence of anti-Brucella abortusantibodies (by the skin test, the agglutination test, and the complement fixation test) and theNramp1genotype (by capillary electrophoresis). Four alleles (Nramp1A, -B, -C, and -D) were detected in the 3′ untranslated region of theNramp1gene. TheBBgenotype was represented among only controls, providing evidence that this genotype confers resistance toBrucella abortus. The monocytes from the BB (resistant) subjects displayed a higher basal level ofNramp1mRNA and a lower number of viable intracellular bacteria than did the monocytes from AA (susceptible) subjects. The higher basal level of the antibacterial protein Nramp1 most probably provides the BB animals with the possibility of controlling bacteria immediately after their entry inside the cell.
Differential Abilities of Capsulated and Noncapsulated
Staphylococcus aureus
Isolates from Diverse
agr
Groups To Invade Mammary Epithelial Cells
Authors: Buzzola Fernanda R. , Alvarez Lucía P. , Tuchscherr Lorena P. N. , Barbagelata María S. , Lattar Santiago M. , Calvinho Luis , Sordelli Daniel O.
Published: December 2006

Staphylococcus aureus
is the bacterium most frequently isolated from milk of bovines with mastitis. Four allelic groups, which interfere with the regulatory activities among the different groups, have been identified in the accessory gene regulator (
agr
) system. The aim of this study was to ascertain the prevalence of the different
agr
groups in capsulated and noncapsulated
S. aureus
bacteria isolated from mastitic bovines in Argentina and whether a given
agr
group was associated with MAC-T cell invasion and in vivo persistence. Eighty-eight percent of the bovine
S. aureus
strains were classified in
agr
group I. The remainder belonged in
agr
groups II, III, and IV (2, 8, and 2%, respectively). By restriction fragment length polymorphism analysis after PCR amplification of the
agr
locus variable region, six
agr
restriction types were identified. All
agr
group I strains presented a unique allele (A/1), whereas strains from groups II, III, and IV exhibited more diversity. Bovine
S. aureus
strains defined as being in
agr
group I (capsulated or noncapsulated) showed significantly increased abilities to be internalized within MAC-T cells, compared with isolates from
agr
groups II, III, and IV.
agr
group II or IV
S. aureus
strains were cleared more efficiently than
agr
group I strains from the murine mammary gland. The results suggest that
agr
group I
S. aureus
strains are more efficiently internalized within epithelial cells and can persist in higher numbers in mammary gland tissue than
S. aureus
strains classified in
agr
group II, III, or IV.
The Early Transcriptional Response of Human Granulocytes to Infection withCandida albicansIs Not Essential for Killing but Reflects Cellular Communications
Authors: Fradin Chantal , Mavor Abigail L. , Weindl Günther , Schaller Martin , Hanke Karin , Kaufmann Stefan H. E. , Mollenkopf Hans , Hube Bernhard
Published: December 2006

Candida albicansis a polymorphic opportunistic fungus that can cause life-threatening systemic infections following hematogenous dissemination in patients susceptible to nosocomial infection. Neutrophils form part of the innate immune response, which is the first line of defense against microbes and is particularly important inC. albicansinfections. To compare the transcriptional response of leukocytes exposed toC. albicans, we investigated the expression of key cytokine genes in polymorphonuclear and mononuclear leukocytes after incubation withC. albicansfor 1 h. Isolated mononuclear cells expressed high levels of genes encoding proinflammatory signaling molecules, whereas neutrophils exhibited much lower levels, similar to those observed in whole blood. The global transcriptional profile of neutrophils was examined by using an immunology-biased human microarray to determine whether different morphological forms or the viability ofC. albicansaltered the transcriptome. Hyphal cells appeared to have the broadest effect, although the most strongly induced genes were regulated independently of morphology or viability. These genes were involved in proinflammatory cell-cell signaling, cell signal transduction, and cell growth. Generally, genes encoding known components of neutrophil granules showed no upregulation at this time point; however, lactoferrin, a well-known candidacidal peptide, was secreted by neutrophils. Addition to inhibitors of RNA or protein de novo synthesis did not influence the killing activity within 30 min. These results support the general notion that neutrophils do not require gene transcription to mount an immediate and direct attack against microbes. However, neutrophils exposed toC. albicansexpress genes involved in communication with other immune cells.
Effect of Site-Specific Mutations in Different Phosphotransfer Domains of the Chemosensory Protein ChpA onPseudomonas aeruginosaMotility
Authors: Leech Andrew J. , Mattick John S.
Published: December 2006

The virulence ofPseudomonas aeruginosaand other surface pathogens involves the coordinate expression of a wide range of virulence determinants, including type IV pili. These surface filaments are important for the colonization of host epithelial tissues and mediate bacterial attachment to, and translocation across, surfaces by a process known as twitching motility. This process is controlled in part by a complex signal transduction system whose central component, ChpA, possesses nine potential sites of phosphorylation, including six histidine-containing phosphotransfer (HPt) domains, one serine-containing phosphotransfer domain, one threonine-containing phosphotransfer domain, and one CheY-like receiver domain. Here, using site-directed mutagenesis, we show that normal twitching motility is entirely dependent on the CheY-like receiver domain and partially dependent on two of the HPt domains. Moreover, under different assay conditions, point mutations in several of the phosphotransfer domains of ChpA give rise to unusual “swarming” phenotypes, possibly reflecting more subtle perturbations in the control ofP. aeruginosamotility that are not evident from the conventional twitching stab assay. Together, these results suggest that ChpA plays a central role in the complex regulation of type IV pilus-mediated motility inP. aeruginosa.
Agrobacterium tumefaciens soxR
Is Involved in Superoxide Stress Protection and Also Directly Regulates Superoxide-Inducible Expression of Itself and a Target Gene
Authors: Eiamphungporn Warawan , Charoenlap Nisanart , Vattanaviboon Paiboon , Mongkolsuk Skorn
Published: December 2006

Inactivation of
Agrobacterium tumefaciens soxR
increases sensitivity to superoxide generators.
soxR
expression is highly induced by superoxide stress and is autoregulated. SoxR also directly regulates the superoxide-inducible expression of atu5152. Taken together, the physiological role of
soxR
and the mechanism by which it regulates expression of target genes make the
A. tumefaciens
SoxR system different from other bacterial systems.
4-Chlorobenzoate Uptake in
Comamonas
sp. Strain DJ-12 Is Mediated by a Tripartite ATP-Independent Periplasmic Transporter
Authors: Chae Jong-Chan , Zylstra Gerben J.
Published: December 2006

The
fcb
gene cluster involved in the hydrolytic dehalogenation of 4-chlorobenzoate is organized in the order
fcbB
-
fcbA
-
fcbT1
-
fcbT2
-
fcbT3
-
fcbC
in
Comamonas
sp. strain DJ-12. The genes are operonic and inducible with 4-chloro-, 4-iodo-, and 4-bromobenzoate. The
fcbT1
,
fcbT2
, and
fcbT3
genes encode a transporter in the secondary TRAP (tripartite ATP-independent periplasmic) family. An
fcbT1T2T3
knockout mutant shows a much slower growth rate on 4-chlorobenzoate compared to the wild type. 4-Chlorobenzoate is transported into the wild-type strain five times faster than into the
fcbT1T2T3
knockout mutant. Transport of 4-chlorobenzoate shows significant inhibition by 4-bromo-, 4-iodo-, and 4-fluorobenzoate and mild inhibition by 3-chlorobenzoate, 2-chlorobenzoate, 4-hydroxybenzoate, 3-hydroxybenzoate, and benzoate. Uptake of 4-chlorobenzoate is significantly inhibited by ionophores which collapse the proton motive force.
The UreEF Fusion Protein Provides a Soluble and Functional Form of the UreF Urease Accessory Protein
Authors: Kim Jong Kyong , Mulrooney Scott B. , Hausinger Robert P.
Published: December 2006

Four accessory proteins (UreD, UreE, UreF, and UreG) are typically required to form the nickel-containing active site in the urease apoprotein (UreABC). Among the accessory proteins, UreD and UreF have been elusive targets for biochemical and structural characterization because they are not overproduced as soluble proteins. Using the best-studied urease system, in which the
Klebsiella aerogenes
genes are expressed in
Escherichia coli
, a translational fusion of
ureE
and
ureF
was generated. The UreEF fusion protein was overproduced as a soluble protein with a convenient tag involving the His-rich region of UreE. The fusion protein was able to form a UreD(EF)G-UreABC complex and to activate urease in vivo, and it interacted with UreD-UreABC in vitro to form a UreD(EF)-UreABC complex. While the UreF portion of UreEF is fully functional, the fusion significantly affected the role of the UreE portion by interrupting its dimerization and altering its metal binding properties compared to those of the wild-type UreE. Analysis of a series of UreEF deletion mutants revealed that the C terminus of UreF is required to form the UreD(EF)G-UreABC complex, while the N terminus of UreF is essential for activation of urease.
Staphylococcus aureus
IsdB Is a Hemoglobin Receptor Required for Heme Iron Utilization
Authors: Torres Victor J. , Pishchany Gleb , Humayun Munir , Schneewind Olaf , Skaar Eric P.
Published: December 2006

The pathogenesis of human infections caused by the gram-positive microbe
Staphylococcus aureus
has been previously shown to be reliant on the acquisition of iron from host hemoproteins. The iron-regulated surface determinant system (Isd) encodes a heme transport apparatus containing three cell wall-anchored proteins (IsdA, IsdB, and IsdH) that are exposed on the staphylococcal surface and hence have the potential to interact with human hemoproteins. Here we report that
S. aureus
can utilize the host hemoproteins hemoglobin and myoglobin, but not hemopexin, as iron sources for bacterial growth. We demonstrate that staphylococci capture hemoglobin on the bacterial surface via IsdB and that inactivation of
isdB
, but not
isdA
or
isdH
, significantly decreases hemoglobin binding to the staphylococcal cell wall and impairs the ability of
S. aureus
to utilize hemoglobin as an iron source. Stable-isotope-tracking experiments revealed removal of heme iron from hemoglobin and transport of this compound into staphylococci. Importantly, mutants lacking
isdB
, but not
isdH
, display a reduction in virulence in a murine model of abscess formation. Thus, IsdB-mediated scavenging of iron from hemoglobin represents an important virulence strategy for
S. aureus
replication in host tissues and for the establishment of persistent staphylococcal infections.
Envying Cinderella and the future of medical enhancements
Authors: Volandes A
Published: December 2006

The medical profession is increasingly absorbing technologies that offer medical care that is more luxury than need. From foot sculpting surgeries to breast and pectoral implants, physicians are increasingly putting patients under the knife to improve on the normal, to make better than well. Medicine’s enthusiasm for absorbing such technologies may corrode the moral priority that it has traditionally held as a profession. Medicine’s moral status declines when it both provides medical luxuries and meets medical needs. Patients without health care will increasingly envy patients who receive healthcare luxuries. Instead, however, of doing away with such luxuries, medicine can institute a pay scheme whereby medical luxuries help subsidise care for those without health care. In this fashion, any feelings of envy are pre-empted and the profession retains its moral status.
The novelArrowsmith, Paul de Kruif (1890–1971) and Jacques Loeb (1859–1924): a literary portrait of “medical science”
Authors: Fangerau H M
Published: December 2006

Shortly after bacteriologist Paul de Kruif had been dismissed from a research position at the Rockefeller Institute for Medical Research, he started contributing to a novel in collaboration with the future Nobel laureate Sinclair Lewis. The novel,Arrowsmith, would become one of the most famous satires on medicine and science. Using de Kruif’s correspondence with his idol Jacques Loeb, this paper describes the many ways in which medical science is depicted inArrowsmith. This article compares the novel with de Kruif’s and Loeb’s biographies, and (1) focuses on the struggles of the main character, Martin Arrowsmith, as an allegory of the institutionalisation of medical research in the US, (2) shows that (influenced by de Kruif) Sinclair’s purpose is to caricaturise scientific work in modern medical research institutions anywhere and (3) shows that the novel depicts a reductionist philosophy of research that seems to contradict the “messiness” of medical practice.
Coeliac disease and primary hyperparathyroidism: an association?
Authors: Maida M J , Praveen E , Crimmins S R , Swift G L
Published: December 2006

Primary hyperparathyroidism may present with non-specific symptoms, and this may be one reason why patients with coeliac disease fail to improve despite compliance with a gluten-free diet. Seven case reports of primary hyperparathyroidism due to sporadic adenoma occurring in a series of 310 patients with coeliac disease are presented, highlighting the importance of looking for this condition in this population group. A prevalence of primary hyperparathyroidism of 2.3% in this series suggests a significant association between hyperparathyroidism and coeliac disease; most studies have indicated a prevalence of 3 in 1000 in the general population, although one study found that it may be as high as 21 in 1000 in women aged 55–75 years. The average age of patients in our series was 59 years and all but one were women. Further studies are needed to establish a possible association between primary hyperparathyroidism and coeliac disease.
NJL INTERACTION DERIVED FROM QCD
Authors: ARBUZOV B. A. , VOLKOV M. K. , ZAITSEV I. V.
Published: December 2006

We apply Bogolubov approach to QCD with two light quarks to demonstrate a spontaneous generation of an effective interaction, leading to the Nambu–Jona-Lasinio model. The resulting theory contains two parameters: average low-energy value of αs and current light quark mass m0. All other low-energy parameters: the pion decay constant, mass of the π-meson, mass of the σ-meson and its width, the constituent quark mass, and the quark condensate are expressed in terms of the two input parameters in satisfactory correspondence to experimental data and chiral phenomenology. For example, in the approximation being used we have for αs = 0.67 and m0 = 20 MeV : fπ = 93 MeV , mπ = 135 MeV , mσ = 492 MeV , Γσ = 574 MeV , mq = 295 MeV , [Formula: see text].
BLACK HOLES, SPACE-FILLING CHAINS AND RANDOM WALKS
Authors: KRAUSE AXEL
Published: December 2006

Many approaches to a semiclassical description of gravity lead to an integer black hole entropy. In four dimensions this implies that the Schwarzschild radius obeys a formula which describes the distance covered by a Brownian random walk. For the higher-dimensional Schwarzschild–Tangherlini black hole, its radius relates similarly to a fractional Brownian walk. We propose a possible microscopic explanation for these random walk structures based on microscopic chains which fill the interior of the black hole.
SIZE AND DIELECTRIC DEPENDENCE OF INTERFACE AND SURFACE OPTICAL PHONONS SPECTRA IN WURTZITE GaN/AlN QUANTUM WELL WIRES
Authors: ZHANG LI
Published: December 2006

By employing the method of electrostatic potential expansion, the interface optical (IO) and surface optical (SO) phonon modes and the corresponding Fröhlich-like electron-phonon interaction Hamiltonian in a Q1D wurtzite cylindrical quantum well wire (QWW) embedded in nonpolar dielectric matrix are derived and studied based on the dielectric continuum model and Loudon's uniaxial crystal model. Numerical calculations for a wurtzite GaN/AlN QWW are mainly focused on the size- and dielectric-dependent IO and SO phonon spectra and electron-IO (SO) phonons coupling functions. Results reveal that, in general, there are two branches of IO phonon modes and one branch of SO mode in the system. The dispersions of the IO and SO modes are obvious only when the radii ratio β and the dielectric constant of nonpolar matrix ∊d is small. The limiting frequencies of IO and SO modes for very large β have been analyzed in depth from both physical and mathematical viewpoints. The reducing behaviors of some modes have been clearly observed. Via the discussion of electrostatic potential spacial distributions of the IO and SO modes, we find that the QWW structures and dielectric constants of nonpolar matrix have little influence on the low-frequency IO mode, but they can greatly affect the potential distributions of high-frequency IO and SO modes. Detailed comparison of the dispersion behaviors of the modes and electron-phonon coupling properties in the Q1D wurtzite QWWs with those in wurtzite QWs and cubic quantum dots has also been made. Furthermore, part of the theoretical results derived in the present paper is consistent with the relatively experimental conclusion.
ANALYSIS OF A VECTOR-HOST MALARIA MODEL WITH IMPULSIVE EFFECT AND INFECTION-AGE
Authors: LIU HELONG , YU JINGYUAN , ZHU GUANGTIAN
Published: December 2006

In this paper, we develop a mathematical model for malaria with impulsive effect and infection-age, and consider the effect of blood transfusion and infected vector transmission. We derive the condition under which malaria goes to extinction.
A Wavelet Packets Approach to Electrocardiograph Baseline Drift Cancellation
Authors: Tinati Mohammad Ali , Mozaffary Behzad
Published: December 2006

Baseline wander elimination is considered a classical problem. In electrocardiography (ECG) signals, baseline drift can influence the accurate diagnosis of heart disease such as ischemia and arrhythmia. We present a wavelet‐transform‐ (WT‐) based search algorithm using the energy of the signal in different scales to isolate baseline wander from the ECG signal. The algorithm computes wavelet packet coefficients and then in each scale the energy of the signal is calculated. Comparison is made and the branch of the wavelet binary tree corresponding to higher energy wavelet spaces is chosen. This algorithm is tested using the data record from MIT/BIH database and excellent results are obtained.
Products of derivations which act as Lie derivations oncommutators of right ideals
Authors: De Filippis V.
Published: December 2006

Let R be a prime ring of characteristic different from 2, I a nonzero right ideal of R, d and δ nonzero derivations of R, and s4(x1, x2, x3, x4) the standard identity of degree 4. If the composition (dδ) is a Lie derivation of [I, I] into R, then either s4(I, I, I, I)I = 0 or δ(I)I = d(I)I = 0.
An identity related to Jordan′s inequality
Authors: Li Jian-Lin
Published: December 2006

The main purpose of this note is to establish an
identity which states that the function sinx/x is a power series of (π2 − 4x2) with positive coefficients for all x ≠ 0. This enable us to obtain a much stronger Jordan′s inequality
than that obtained before.
Tauberian conditions for a general limitable method
Authors: Canak İbrahim , Totur Ümit
Published: December 2006

Let (un)
be a sequence of real numbers, L
an additive
limitable method with some property, and and
different spaces of sequences related to each other.
We prove that if the classical control modulo of the oscillatory
behavior of (un) in is a Tauberian condition
for L, then the general control modulo of the oscillatory
behavior of integer order m of (un)
in or
is also a Tauberian condition for L.
MUC1/X Protein Immunization Enhances cDNA Immunization in Generating Anti-MUC1 α/β Junction Antibodies that Target Malignant Cells
Authors: Rubinstein Daniel B. , Karmely Maya , Ziv Ravit , Benhar Itai , Leitner Orit , Baron Shoshana , Katz Ben-Zion , Wreschner Daniel H.
Published: December 2006

MUC1 has generated considerable interest as a tumor marker and potential target for tumor killing. To date, most antibodies against MUC1 recognize epitopes within the highly immunogenic α chain tandem repeat array. A major shortcoming of such antibodies is that the MUC1 α chain is shed into the peripheral circulation, sequesters circulating antitandem repeat array antibodies, and limits their ability to even reach targeted MUC1-expressing cells. Antibodies recognizing MUC1 epitopes tethered to the cell surface would likely be more effective. MUC1 α subunit binding the membrane-tethered β subunit provides such an epitope. By use of a novel protocol entailing immunization with cDNA encoding full-length MUC1 (MUC1/TM) followed by boosting with the alternatively spliced MUC1/X isoform from which the tandem repeat array has been deleted, we generated monoclonal antibodies, designated DMC209, which specifically bind the MUC1 α/β junction. DMC209 is exquisitely unique for this site; amino acid mutations, which abrogate MUC1 cleavage, also abrogate DMC209 binding. Additionally, DMC209 specifically binds the MUC1 α/β junction on full-length MUC1/TM expressed by breast and ovarian cancer cell lines and on freshly obtained, unmanipulated MUC1-positive malignant plasma cells of multiple myeloma. DMC209 is likely to have clinical application by targeting MUC1-expressing cells directly and as an immunotoxin conjugate. Moreover, the novel immunization procedure used in generating DMC209 can be used to generate additional anti-MUC1 α/β junction antibodies, which may, analogously to Herceptin, have cytotoxic activity. Lastly, sequential immunization with MUC1/TM cDNA acting as a nonspecific adjuvant followed by protein of interest may prove to be a generalizable method to yield high-titer specific antibodies. (Cancer Res 2006; 66(23): 11247-53)
Mesenchymal-to-Epithelial Transition Facilitates Bladder Cancer Metastasis: Role of Fibroblast Growth Factor Receptor-2
Authors: Chaffer Christine L. , Brennan Janelle P. , Slavin John L. , Blick Tony , Thompson Erik W. , Williams Elizabeth D.
Published: December 2006

Epithelial-to-mesenchymal transition (EMT) increases cell migration and invasion, and facilitates metastasis in multiple carcinoma types, but belies epithelial similarities between primary and secondary tumors. This study addresses the importance of mesenchymal-to-epithelial transition (MET) in the formation of clinically significant metastasis. The previously described bladder carcinoma TSU-Pr1 (T24) progression series of cell lines selected in vivo for increasing metastatic ability following systemic seeding was used in this study. It was found that the more metastatic sublines had acquired epithelial characteristics. Epithelial and mesenchymal phenotypes were confirmed in the TSU-Pr1 series by cytoskeletal and morphologic analysis, and by performance in a panel of in vitro assays. Metastatic ability was examined following inoculation at various sites. Epithelial characteristics associated with dramatically increased bone and soft tissue colonization after intracardiac or intratibial injection. In contrast, the more epithelial sublines showed decreased lung metastases following orthotopic inoculation, supporting the concept that EMT is important for the escape of tumor cells from the primary tumor. We confirmed the overexpression of the IIIc subtype of multiple fibroblast growth factor receptors (FGFR) through the TSU-Pr1 series, and targeted abrogation of FGFR2IIIc reversed the MET and associated functionality in this system and increased survival following in vivo inoculation in severe combined immunodeficient mice. This model is the first to specifically model steps of the latter part of the metastatic cascade in isogenic cell lines, and confirms the suspected role of MET in secondary tumor growth. (Cancer Res 2006; 66(23): 11271-8)
Cell Adhesion Molecule L1 Disrupts E-Cadherin-Containing Adherens Junctions and Increases Scattering and Motility of MCF7 Breast Carcinoma Cells
Authors: Shtutman Michael , Levina Elina , Ohouo Patrice , Baig Mirza , Roninson Igor B.
Published: December 2006

The first steps of invasion and metastasis include the dissociation of adherens junctions and the induction of migratory phenotype, through a program that resembles epithelial-mesenchymal transition (EMT). The L1 cell adhesion molecule, which is normally found primarily in the brain, was recently shown to be expressed in different types of cancer and to have tumor-promoting activity. We now find that L1 mediates EMT-like events in MCF7 breast carcinoma cells. MCF7 predominantly expresses the nonneuronal isoform of L1, as do 16 of 17 other cell lines derived from different types of cancer. L1 protein expression in MCF7 cells, which form E-cadherin-containing adherens junctions, is inversely related to cell density. Analysis of MCF7 cells with overexpression or knockdown of nonneuronal L1 isoform revealed that L1 expression leads to the disruption of adherens junctions and increases β-catenin transcriptional activity. As a result, L1 expression promotes the scattering of epithelial cells from compact colonies. Expression of the full-length L1 protein, but not of its soluble extracellular moiety, increases the motility of the MCF7 epithelial monolayer in a wound-healing assay, in which L1 expression is preferentially observed and required in cells leading the movement of the monolayer. Based on these results, we propose a model for the role of L1 as a trigger of EMT-like events in transformed epithelial cells. (Cancer Res 2006; 66(23): 11370-80)
Cyclooxygenase-2 and Colorectal Cancer Chemoprevention: The β-Catenin Connection
Authors: Castellone Maria Domenica , Teramoto Hidemi , Gutkind J. Silvio
Published: December 2006

Colorectal cancer poses a major clinical challenge in the developed world where this disease is common. Recent findings suggest that the prostaglandin E2, the proinflammatory product of elevated cyclooxygenase-2 activity in colon cancer, stimulates cancer cell growth through a G protein–dependent signaling pathway coupling the prostaglandin EP2 receptor to β-catenin control. These findings provide new insights into the molecular framework needed to evaluate chemopreventive strategies for colorectal cancer. (Cancer Res 2006; 66(23): 11085-8)
Transcriptional Profiling Identifies Cyclin D1 as a Critical Downstream Effector of Mutant Epidermal Growth Factor Receptor Signaling
Authors: Kobayashi Susumu , Shimamura Takeshi , Monti Stefano , Steidl Ulrich , Hetherington Christopher J. , Lowell April M. , Golub Todd , Meyerson Matthew , Tenen Daniel G. , Shapiro Geoffrey I. , Halmos Balázs
Published: December 2006

Activating mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain determine responsiveness to EGFR tyrosine kinase inhibitors in patients with advanced non–small cell lung cancer (NSCLC). The modulation of transcriptional pathways by mutant EGFR signaling is not fully understood. Previously, we and others identified a single base pair change leading to a threonine to methionine (T790M) amino acid alteration in the ATP-binding pocket of the EGFR as a common mechanism of acquired resistance. The gefitinib-resistant, T790M-mutant H1975 NSCLC cell line undergoes prominent growth arrest and apoptosis when treated with the irreversible EGFR inhibitor, CL-387,785. We did a transcriptional profiling study of mutant EGFR target genes that are differentially expressed in the “resistant” gefitinib-treated and the “sensitive” CL387,785-treated H1975 cells to identify the pivotal transcriptional changes in NSCLC with EGFR-activating mutations. We identified a small subset of early gene changes, including significant reduction of cyclin D1 as a result of EGFR inhibition by CL-387,785 but not by gefitinib. The reduction in cyclin D1 transcription was associated with subsequent suppression of E2F-responsive genes, consistent with proliferation arrest. Furthermore, cyclin D1 expression was higher in EGFR-mutant lung cancer cells compared with cells with wild-type EGFR. EGFR-mutant cells were routinely sensitive to the cyclin-dependent kinase inhibitor flavopiridol, confirming the functional relevance of the cyclin D axis. These studies suggest that cyclin D1 may contribute to the emergence of EGFR-driven tumorigenesis and can be an alternative target of therapy. (Cancer Res 2006; 66(23): 11389-98)
Isolation of Germ Cells from Leukemia and Lymphoma Cells in a Human In vitro Model: Potential Clinical Application for Restoring Human Fertility after Anticancer Therapy
Authors: Fujita Kazutoshi , Tsujimura Akira , Miyagawa Yasushi , Kiuchi Hiroshi , Matsuoka Yasuhiro , Takao Tetsuya , Takada Shingo , Nonomura Norio , Okuyama Akihiko
Published: December 2006

More than 70% of patients survive childhood cancer, but chemotherapy and radiation therapy may cause irreversible impairment of spermatogenesis. To treat infertility secondary to anticancer treatment for childhood cancer, we have developed a procedure to isolate germ cells from leukemic mice by fluorescence-activated cell sorting with two surface markers, and transplantation of isolated germ cells successfully restored fertility without inducing leukemia. In the present study, we analyzed human germ cells and human malignant cells, including five leukemia cell lines and three lymphoma cell lines, by fluorescence-activated cell sorting with antibodies against MHC class I and CD45. Testicular specimens were obtained from a patient who underwent surgery for testicular rupture. In the high forward scatter and low side scatter region, no malignant cells were found in the MHC class I-negative and CD45-negative fraction (the germ cell fraction), with the exception of K562 cells. A total of 39.2% of the germ cells were found in the germ cell fraction. A total of 1.45% of K562 cells were found in the germ cell fraction. Treatment with IFNγ induced the expression of MHC class I on K562 cells but not on germ cells and made it possible to isolate germ cells from K562 cells. In conclusion, we isolated human germ cells from malignant cells with two surface markers after treatment with IFNγ. Immunophenotyping for each patient will be necessary before isolation and induction of surface marker will be clinically applicable. (Cancer Res 2006; 66(23): 11166-71)