bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Expression Vector for Secretion of Cecropin AD in
Bacillus subtilis
with Enhanced Antimicrobial Activity
Authors: Chen Xiang , Zhu Faming , Cao Yunhe , Qiao Shiyan
Published: June 2009

Cecropin AD, a chimeric antimicrobial peptide obtained from cecropins, is effective at killing specific microorganisms. However, a highly efficient expression system is still needed to allow for commercial application of cecropin AD. For the exogenous expression of cecropin AD, we fused the cecropin AD gene with a small ubiquitin-like modifier (SUMO) gene and a signal peptide of SacB, while a
Bacillus subtilis
expression system was constructed based on
Bacillus subtilis
cells genetically modified by the introduction of an operon including an isopropyl-β-
d
-thiogalactopyranoside (IPTG)-inducible Spac promoter, a signal peptide of
amyQ
, and the SUMO protease gene. The recombinant cecropin AD was expressed, and 30.6 mg of pure recombinant protein was purified from 1 liter of culture supernatant. The purified cecropin AD displayed antimicrobial activity against some pathogens, such as
Staphylococcus aureus
and
Escherichia coli
, and was especially effective toward
Staphylococcus aureus
, with MICs of <0.05 μM (0.2 μg/ml). Stability analysis results showed that the activity of cecropin AD was not influenced by temperatures as high as 55°C for 20 min; however, temperatures above 85°C (for 20 min) decreased the antimicrobial activity of cecropin AD. Varying the pH from 4.0 to 9.0 did not appear to affect the activity of cecropin AD, but some loss of potency was observed at pH values lower than pH 4.0. Under the challenge of several proteases (proteinase K, trypsin, and pepsin), cecropin AD maintained functional activity. The results indicated that the recombinant product expressed by the designed
Bacillus subtilis
expression system was a potent antimicrobial agent and could be applied to control infectious diseases of farm animals or even humans.
Potent Activity of a Nucleoside Reverse Transcriptase Inhibitor, 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine, against Human Immunodeficiency Virus Type 1 Infection in a Model Using Human Peripheral Blood Mononuclear Cell-Transplanted NOD/SCID Janus Kinase 3 Knockout Mice
Authors: Hattori Shinichiro , Ide Kazuhiko , Nakata Hirotomo , Harada Hideki , Suzu Shinya , Ashida Noriyuki , Kohgo Satoru , Hayakawa Hiroyuki , Mitsuya Hiroaki , Okada Seiji
Published: June 2009

4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA), a recently discovered nucleoside reverse transcriptase inhibitor, exhibits activity against a wide spectrum of wild-type and multidrug-resistant clinical human immunodeficiency virus type 1 (HIV-1) isolates (50% effective concentration, 0.0001 to 0.001 μM). In the present study, we used human peripheral blood mononuclear cell-transplanted, HIV-1-infected NOD/SCID/Janus kinase 3 knockout mice for in vivo evaluation of the anti-HIV activity of EFdA. Administration of EFdA decreased the replication and cytopathic effects of HIV-1 without identifiable adverse effects. In phosphate-buffered saline (PBS)-treated mice, the CD4
+
/CD8
+
cell ratio in the spleen was low (median, 0.04; range, 0.02 to 0.49), while that in mice receiving EFdA was increased (median, 0.65; range, 0.57 to 1.43). EFdA treatment significantly suppressed the amount of HIV-1 RNA (median of 9.0 × 10
2
copies/ml [range, 8.1 × 10
2
to 1.1 × 10
3
copies/ml] versus median of 9.9 × 10
4
copies/ml [range, 8.1 × 10
2
to 1.1 × 10
3
copies/ml];
P
< 0.001), the p24 level in plasma (2.5 × 10
3
pg/ml [range, 8.2 × 10
2
to 5.6 × 10
3
pg/ml] versus 2.8 × 10
2
pg/ml [range, 8.2 × 10
1
to 6.3 × 10
2
pg/ml];
P
< 0.001), and the percentage of p24-expressing cells in the spleen (median of 1.90% [range, 0.33% to 3.68%] versus median of 0.11% [range, 0.00% to 1.00%];
P
= 0.003) in comparison with PBS-treated mice. These data suggest that EFdA is a promising candidate for a new age of HIV-1 chemotherapy and should be developed further as a potential therapy for individuals with multidrug-resistant HIV-1 variants.
Biofilm Formation and Effect of Caspofungin on Biofilm Structure of
Candida
Species Bloodstream Isolates
Authors: Ferreira J. A. G. , Carr J. H. , Starling C. E. F. , de Resende M. A. , Donlan R. M.
Published: June 2009

Candida
biofilms are microbial communities, embedded in a polymeric matrix, growing attached to a surface, and are highly recalcitrant to antimicrobial therapy. These biofilms exhibit enhanced resistance against most antifungal agents except echinocandins and lipid formulations of amphotericin B. In this study, biofilm formation by different
Candida
species, particularly
Candida albicans
,
C. tropicalis
, and
C. parapsilosis
, was evaluated, and the effect of caspofungin (CAS) was assessed using a clinically relevant in vitro model system. CAS displayed in vitro activity against
C. albicans
and
C. tropicalis
cells within biofilms. Biofilm formation was evaluated after 48 h of antifungal drug exposure, and the effects of CAS on preformed
Candida
species biofilms were visualized using scanning electron microscopy (SEM). Several species-specific differences in the cellular morphologies associated with biofilms were observed. Our results confirmed the presence of paradoxical growth (PG) in
C. albicans
and
C. tropicalis
biofilms in the presence of high CAS concentrations. These findings were also confirmed by SEM analysis and were associated with the metabolic activity obtained by biofilm susceptibility testing. Importantly, these results suggest that the presence of atypical, enlarged, conical cells could be associated with PG and with tolerant cells in
Candida
species biofilm populations. The clinical implications of these findings are still unknown.
In Vitro and In Vivo Properties of Dihydrophthalazine Antifolates, a Novel Family of Antibacterial Drugs
Authors: Caspers Patrick , Bury Luc , Gaucher Bérangère , Heim Jutta , Shapiro Stuart , Siegrist Sibylle , Schmitt-Hoffmann Anne , Thenoz Laure , Urwyler Heinrich
Published: June 2009

Racemic 2,4-diaminopyrimidine dihydrophthalazine derivatives BAL0030543, BAL0030544, and BAL0030545 exhibited low in vitro MICs toward small, selected panels of
Enterococcus faecalis
,
Enterococcus faecium
,
Streptococcus pneumoniae
,
Moraxella catarrhalis
, and
Mycobacterium avium
, though the compounds were less active against
Haemophilus influenzae
. The constellation of dihydrofolate reductases (DHFRs) present in 20 enterococci and 40 staphylococci was analyzed and correlated with the antibacterial activities of the dihydrophthalazines and trimethoprim. DHFRs encoded by
dfrB
,
dfrA
(S1 isozyme),
dfrE
, and
folA
were susceptible to the dihydrophthalazines, whereas DHFRs encoded by
dfrG
(S3 isozyme) and
dfrF
were not. Studies with the separated enantiomers of BAL0030543, BAL0030544, and BAL0030545 revealed preferential inhibition of susceptible DHFRs by the (
R
)-enantiomers. BAL0030543, BAL0030544, and BAL0030545 were well tolerated by mice during 5- and 10-day oral toxicity studies at doses of up to 400 mg/kg of body weight. Using a nonoptimized formulation, the dihydrophthalazines displayed acceptable oral bioavailabilities in mice, and efficacy studies with a septicemia model of mice infected with trimethoprim-resistant, methicillin-resistant
Staphylococcus aureus
gave 50% effective dose values in the range of 1.6 to 6.25 mg/kg.
Effect of
Candida glabrata FKS1
and
FKS2
Mutations on Echinocandin Sensitivity and Kinetics of 1,3-β-
d
-Glucan Synthase: Implication for the Existing Susceptibility Breakpoint
Authors: Garcia-Effron Guillermo , Lee Samuel , Park Steven , Cleary John D. , Perlin David S.
Published: June 2009

Thirteen
Candida glabrata
strains harboring a range of mutations in hot spot regions of
FKS1
and
FKS2
were studied. The mutations were linked to an echinocandin reduced susceptibility phenotype. Sequence alignments showed that 11 out of the 13 mutants harbored a mutation in
FKS1
or
FKS2
not previously implicated in echinocandin reduced susceptibility in
C. glabrata
. A detailed kinetic characterization demonstrated that amino acid substitutions in Fks1p and Fks2p reduced drug sensitivity in mutant 1,3-β-
d
-glucan synthase by 2 to 3 log orders relative to that in wild-type enzyme. These mutations were also found to reduce the catalytic efficiency of the enzyme (
V
max
) and to influence the relative expression of
FKS
genes. In view of the association of
FKS
mutations and reduced susceptibility of 1,3-β-
d
-glucan synthase, an evaluation of the new CLSI echinocandin susceptibility breakpoint was conducted. Only 3 of 13 resistant
fks
mutants (23%) were considered anidulafungin or micafungin nonsusceptible (MIC > 2 μg/ml) by this criterion. In contrast, most
fks
mutants (92%) exceeded a MIC of >2 μg/ml with caspofungin. However, when MIC determinations were performed in the presence of 50% serum, all
C. glabrata fks
mutants showed MICs of ≥2 μg/ml for the three echinocandin drugs. As has been observed with
Candida albicans
, the kinetic inhibition parameter 50% inhibitory concentration may be a better predictor of
FKS
-mediated resistance. Finally, the close association between
FKS1/FKS2
hot spot mutations provides a basis for understanding echinocandin resistance in
C. glabrata
.
Widespread Distribution of Poribacteria in Demospongiae
Authors: Lafi Feras F. , Fuerst John A. , Fieseler Lars , Engels Cecilia , Goh Winnie Wei Ling , Hentschel Ute
Published: June 2009

Poribacteria were found in nine sponge species belonging to six orders of Porifera from three oceans. Phylogenetic analysis revealed four distinct poribacterial clades, which contained organisms obtained from several different geographic regions, indicating that the distribution of poribacteria is cosmopolitan. Members of divergent poribacterial clades were also found in the same sponge species in three different sponge genera.
Rhodococcus rhodochrous
DSM 43269 3-Ketosteroid 9α-Hydroxylase, a Two-Component Iron-Sulfur-Containing Monooxygenase with Subtle Steroid Substrate Specificity
Authors: Petrusma M. , Dijkhuizen L. , van der Geize R.
Published: June 2009

This paper reports the biochemical characterization of a purified and reconstituted two-component 3-ketosteroid 9α-hydroxylase (KSH). KSH of
Rhodococcus rhodochrous
DSM 43269, consisting of a ferredoxin reductase (KshB) and a terminal oxygenase (KshA), was heterologously expressed in
Escherichia coli. E. coli
cell cultures, expressing both KshA and KshB, converted 4-androstene-3,17-dione (AD) into 9α-hydroxy-4-AD (9OHAD) with a >60% molar yield over 48 h of incubation. Coexpression and copurification were critical to successfully obtain pure and active KSH. Biochemical analysis revealed that the flavoprotein KshB is an NADH-dependent reductase using flavin adenine dinucleotide as a cofactor. Reconstitution experiments confirmed that KshA, KshB, and NADH are essential for KSH activity with steroid substrates. KSH hydroxylation activity was inhibited by several divalent metal ions, especially by zinc. The reconstituted KSH displayed subtle steroid substrate specificity; a range of 3-ketosteroids, i.e., 5α-Η, 5β-Η, Δ1, and Δ4 steroids, could act as KSH substrates, provided that they had a short side chain. The formation of 9OHAD from AD by KSH was confirmed by liquid chromatography-mass spectrometry analysis and by the specific enzymatic conversion of 9OHAD into 3-hydroxy-9,10-secoandrost-1,3,5(10)-triene-9,17-dione using 3-ketosteroid Δ1-dehydrogenase. Only a single KSH is encoded in the genome of the human pathogen
Mycobacterium tuberculosis
H37Rv, shown to be important for survival in macrophages. Since no human KSH homolog exists, the
M. tuberculosis
enzyme may provide a novel target for treatment of tuberculosis. Detailed knowledge about the biochemical properties of KSH thus is highly relevant in the research fields of biotechnology and medicine.
Assessment of Bias Associated with Incomplete Extraction of Microbial DNA from Soil
Authors: Feinstein Larry M. , Sul Woo Jun , Blackwood Christopher B.
Published: June 2009

DNA extraction bias is a frequently cited but poorly understood limitation of molecular characterizations of environmental microbial communities. To assess the bias of a commonly used soil DNA extraction kit, we varied the cell lysis protocol and conducted multiple extractions on subsamples of clay, sand, and organic soils. DNA, as well as bacterial and fungal ribosomal gene copies as measured by quantitative PCR, continued to be isolated in successive extractions. When terminal restriction fragment length polymorphism was used, a significant shift in community composition due to extraction bias was detected for bacteria but not for fungi. Pyrosequencing indicated that the relative abundances of sequences from rarely cultivated groups such asAcidobacteria,Gemmatimonades, andVerrucomicrobiawere higher in the first extraction than in the sixth but that the reverse was true forProteobacteriaandActinobacteria. This suggests that the well-known phylum-level bacterial cultivation bias may be partially exaggerated by DNA extraction bias. We conclude that bias can be adequately reduced in many situations by pooling three successive extractions, and additional measures should be considered when divergent soil types are compared or when comprehensive community analysis is necessary.
Relative Diversity and Community Structure of Ciliates in Stream Biofilms According to Molecular and Microscopy Methods
Authors: Dopheide Andrew , Lear Gavin , Stott Rebecca , Lewis Gillian
Published: June 2009

Ciliates are an important component of aquatic ecosystems, acting as predators of bacteria and protozoa and providing nutrition for organisms at higher trophic levels. Understanding of the diversity and ecological role of ciliates in stream biofilms is limited, however. Ciliate diversity in biofilm samples from four streams subject to different impacts by human activity was assessed using microscopy and terminal restriction fragment length polymorphism (T-RFLP) analysis of 18S rRNA sequences. Analysis of 3′ and 5′ terminal fragments yielded very similar estimates of ciliate diversity. The diversity detected using microscopy was consistently lower than that suggested by T-RFLP analysis, indicating the existence of genetic diversity not apparent by morphological examination. Microscopy and T-RFLP analyses revealed similar relative trends in diversity between different streams, with the lowest level of biofilm-associated ciliate diversity found in samples from the least-impacted stream and the highest diversity in samples from moderately to highly impacted streams. Multivariate analysis provided evidence of significantly different ciliate communities in biofilm samples from different streams and seasons, particularly between a highly degraded urban stream and less impacted streams. Microscopy and T-RFLP data both suggested the existence of widely distributed, resilient biofilm-associated ciliates as well as ciliate taxa restricted to sites with particular environmental conditions, with cosmopolitan taxa being more abundant than those with restricted distributions. Differences between ciliate assemblages were associated with water quality characteristics typical of urban stream degradation and may be related to factors such as nutrient availability and macroinvertebrate communities. Microscopic and molecular techniques were considered to be useful complementary approaches for investigation of biofilm ciliate communities.
Identification and Characterization of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase from
Bacillus megaterium
Authors: Chen Hui-Ju , Pan Shih-Chuan , Shaw Gwo-Chyuan
Published: June 2009

A gene that codes for a novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase, designated PhaZ1, has been identified in the genome of
Bacillus megaterium
. A native PHB (nPHB) granule-binding assay showed that purified soluble PhaZ1 had strong affinity for nPHB granules. Turbidimetric analyses revealed that PhaZ1 could rapidly degrade nPHB granules in vitro without the need for protease pretreatment of the granules to remove surface proteins. Notably, almost all the final hydrolytic products produced from the in vitro degradation of nPHB granules by PhaZ1 were 3-hydroxybutyric acid (3HB) monomers. Unexpectedly, PhaZ1 could also hydrolyze denatured semicrystalline PHB, with the generation of 3HB monomers. The disruption of the
phaZ1
gene significantly affected intracellular PHB mobilization during the PHB-degrading stage in
B. megaterium
, as demonstrated by transmission electron microscopy and the measurement of the PHB content. These results indicate that PhaZ1 is functional in intracellular PHB mobilization in vivo. Some of these features, which are in striking contrast with those of other known nPHB granule-degrading PhaZs, may provide an advantage for
B. megaterium
PhaZ1 in fermentative production of the biotechnologically valuable chiral compound (
R
)-3HB.
RNA Colony Blot Hybridization Method for Enumeration of Culturable
Vibrio cholerae
and
Vibrio mimicus
Bacteria
Authors: Grim Christopher J. , Zo Young-Gun , Hasan Nur A. , Ali Afsar , Chowdhury Wasimul B. , Islam Atiqul , Rashid Mohammed H. , Alam Munirul , Morris J. Glenn , Huq Anwar , Colwell Rita R.
Published: June 2009

A species-specific RNA colony blot hybridization protocol was developed for enumeration of culturable
Vibrio cholerae
and
Vibrio mimicus
bacteria in environmental water samples. Bacterial colonies on selective or nonselective plates were lysed by sodium dodecyl sulfate, and the lysates were immobilized on nylon membranes. A fluorescently labeled oligonucleotide probe targeting a phylogenetic signature sequence of 16S rRNA of
V. cholerae
and
V. mimicus
was hybridized to rRNA molecules immobilized on the nylon colony lift blots. The protocol produced strong positive signals for all colonies of the 15 diverse
V. cholerae
-
V. mimicus
strains tested, indicating 100% sensitivity of the probe for the targeted species. For visible colonies of 10 nontarget species, the specificity of the probe was calculated to be 90% because of a weak positive signal produced by
Grimontia
(
Vibrio
)
hollisae
, a marine bacterium. When both the sensitivity and specificity of the assay were evaluated using lake water samples amended with a bioluminescent
V. cholerae
strain, no false-negative or false-positive results were found, indicating 100% sensitivity and specificity for culturable bacterial populations in freshwater samples when
G. hollisae
was not present. When the protocol was applied to laboratory microcosms containing
V. cholerae
attached to live copepods, copepods were found to carry approximately 10,000 to 50,000 CFU of
V. cholerae
per copepod. The protocol was also used to analyze pond water samples collected in an area of cholera endemicity in Bangladesh over a 9-month period. Water samples collected from six ponds demonstrated a peak in abundance of total culturable
V. cholerae
bacteria 1 to 2 months prior to observed increases in pathogenic
V. cholerae
and in clinical cases recorded by the area health clinic. The method provides a highly specific and sensitive tool for monitoring the dynamics of
V. cholerae
in the environment. The RNA blot hybridization protocol can also be applied to detection of other gram-negative bacteria for taxon-specific enumeration.
Development of a Cell Culture Method To Isolate and Enrich
Salmonella enterica
Serotype Enteritidis from Shell Eggs for Subsequent Detection by Real-Time PCR
Authors: Day J. B. , Basavanna U. , Sharma S. K.
Published: June 2009

Salmonella enterica
serotype Enteritidis is a major cause of nontyphoidal salmonellosis from ingestion of contaminated raw or undercooked shell eggs. Current techniques used to identify
Salmonella
serotype Enteritidis in eggs are extremely laborious and time-consuming. In this study, a novel eukaryotic cell culture system was combined with real-time PCR analysis to rapidly identify
Salmonella
serotype Enteritidis in raw shell eggs. The system was compared to the standard microbiological method of the International Organization for Standardization (Anonymous, Microbiology of food and animal feeding stuffs—horizontal method for the detection of
Salmonella
, 2002). The novel technique utilizes a mouse macrophage cell line (RAW 264.7) as the host for the isolation and intracellular replication of
Salmonella
serotype Enteritidis. Exposure of macrophages to
Salmonella
serotype Enteritidis-contaminated eggs results in uptake and intracellular replication of the bacterium, which can subsequently be detected by real-time PCR analysis of the DNA released after disruption of infected macrophages. Macrophage monolayers were exposed to eggs contaminated with various quantities of
Salmonella
serotype Enteritidis. As few as 10 CFU/ml was detected in cell lysates from infected macrophages after 10 h by real-time PCR using primer and probe sets specific for DNA segments located on the
Salmonella
serotype Enteritidis genes
sefA
and
orgC
.
Salmonella
serotype Enteritidis could also be distinguished from other non-serogroup D
Salmonella
serotypes by using the
sefA
- and
orgC
-specific primer and probe sets. Confirmatory identification of
Salmonella
serotype Enteritidis in eggs was also achieved by isolation of intracellular bacteria from lysates of infected macrophages on xylose lysine deoxycholate medium. This method identifies
Salmonella
serotype Enteritidis from eggs in less than 10 h compared to the more than 5 days required for the standard reference microbiological method of the International Organization for Standardization (Microbiology of food and animal feeding stuffs—horizontal method for the detection of
Salmonella
, 2002).
Sulfur Isotope Enrichment during Maintenance Metabolism in the Thermophilic Sulfate-Reducing Bacterium
Desulfotomaculum putei
Authors: Davidson Mark M. , Bisher M. E. , Pratt Lisa M. , Fong Jon , Southam Gordon , Pfiffner Susan M. , Reches Z. , Onstott Tullis C.
Published: June 2009

Values of Δ
34
S (

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({=}{\delta}^{34}S_{HS}{-}{\delta}^{34}S_{SO_{4}}\) \end{document}

, where δ
34
S
HS
and

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\delta}^{34}S_{SO_{4}}\) \end{document}

indicate the differences in the isotopic compositions of the HS
−
and SO
4
2−
in the eluent, respectively) for many modern marine sediments are in the range of −55 to −75‰, much greater than the −2 to −46‰ ε
34
S (kinetic isotope enrichment) values commonly observed for microbial sulfate reduction in laboratory batch culture and chemostat experiments. It has been proposed that at extremely low sulfate reduction rates under hypersulfidic conditions with a nonlimited supply of sulfate, isotopic enrichment in laboratory culture experiments should increase to the levels recorded in nature. We examined the effect of extremely low sulfate reduction rates and electron donor limitation on S isotope fractionation by culturing a thermophilic, sulfate-reducing bacterium,
Desulfotomaculum putei
, in a biomass-recycling culture vessel, or “retentostat.” The cell-specific rate of sulfate reduction and the specific growth rate decreased progressively from the exponential phase to the maintenance phase, yielding average maintenance coefficients of 10
−16
to 10
−18
mol of SO
4
cell
−1
h
−1
toward the end of the experiments. Overall S mass and isotopic balance were conserved during the experiment. The differences in the δ
34
S values of the sulfate and sulfide eluting from the retentostat were significantly larger, attaining a maximum Δ
34
S of −20.9‰, than the −9.7‰ observed during the batch culture experiment, but differences did not attain the values observed in marine sediments.
Functional Analysis of
cis
- and
trans
-Acting Elements of the
Candida albicans CDR2
Promoter with a Novel Promoter Reporter System
Authors: Coste Alix T. , Crittin Jérôme , Bauser Christopher , Rohde Bettina , Sanglard Dominique
Published: June 2009

Azole resistance in
Candida albicans
can be mediated by the upregulation of the ATP binding cassette transporter genes
CDR1
and
CDR2
. Both genes are regulated by a
cis
-acting element called the drug-responsive element (DRE), with the consensus sequence 5′-CGGAWATCGGATATTTTTTT-3′, and the transcription factor Tac1p. In order to analyze in detail the DRE sequence necessary for the regulation of
CDR1
and
CDR2
and properties of
TAC1
alleles, a one-hybrid system was designed. This system is based on a

P
(CDR2)
-HIS3

reporter system in which complementation of histidine auxotrophy can be monitored by activation of the reporter system by
CDR2
-inducing drugs such as estradiol. Our results show that most of the modifications within the DRE, but especially at the level of CGG triplets, strongly reduce
CDR2
expression. The
CDR2
DRE was replaced by putative DREs deduced from promoters of coregulated genes (
CDR1
,
RTA3
, and
IFU5
). Surprisingly, even if Tac1p was able to bind these putative DREs, as shown by chromatin immunoprecipitation, those from
RTA3
and
IFU5
did not functionally replace the
CDR2
DRE. The one-hybrid system was also used for the identification of gain-of-function (GOF) mutations either in
TAC1
alleles from clinical
C. albicans
isolates or inserted in TAC1 wild-type alleles by random mutagenesis. In all, 17 different GOF mutations were identified at 13 distinct positions. Five of them (G980E, N972D, A736V, T225A, and N977D) have already been described in clinical isolates, and four others (G980W, A736T, N972S, and N972I) occurred at already-described positions, thus suggesting that GOF mutations can occur in a limited number of positions in Tac1p. In conclusion, the one-hybrid system developed here is rapid and powerful and can be used for characterization of
cis
- and
trans
-acting elements in
C. albicans
.
Amino Acid Changes in Elongation Factor Tu of
Mycoplasma pneumoniae
and
Mycoplasma genitalium
Influence Fibronectin Binding
Authors: Balasubramanian Sowmya , Kannan T. R. , Hart P. John , Baseman Joel B.
Published: June 2009

Mycoplasma pneumoniae
and
Mycoplasma genitalium
are closely related organisms that cause distinct clinical manifestations and possess different tissue predilections despite their high degree of genome homology. We reported earlier that surface-localized
M. pneumoniae
elongation factor Tu (EF-Tu
Mp
) mediates binding to the extracellular matrix component fibronectin (Fn) through the carboxyl region of EF-Tu. In this study, we demonstrate that surface-associated
M. genitalium
EF-Tu (EF-Tu
Mg
), in spite of sharing 96% identity with EF-Tu
Mp
, does not bind Fn. We utilized this finding to identify the essential amino acids of EF-Tu
Mp
that mediate Fn interactions by generating modified recombinant EF-Tu proteins with amino acid changes corresponding to those of EF-Tu
Mg
. Amino acid changes in serine 343, proline 345, and threonine 357 were sufficient to significantly reduce the Fn binding of EF-Tu
Mp
. Synthetic peptides corresponding to this region of EF-Tu
Mp
(EF-Tu
Mp
340-358) blocked both recombinant EF-Tu
Mp
and radiolabeled
M. pneumoniae
cell binding to Fn. In contrast, EF-Tu
Mg
340-358 peptides exhibited minimal blocking activity, reinforcing the specificity of EF-Tu-Fn interactions as mediators of microbial colonization and tissue tropism.
Injection of
Staphylococcus aureus
EDIN by the
Bacillus anthracis
Protective Antigen Machinery Induces Vascular Permeability
Authors: Rolando Monica , Munro Patrick , Stefani Caroline , Auberger Patrick , Flatau Gilles , Lemichez Emmanuel
Published: June 2009

Systemic injection of
Bacillus anthracis
lethal toxin (LT) produces vascular leakage and animal death. Recent studies suggest that LT triggers direct endothelial cell cytotoxicity that is responsible for the vascular leakage. LT is composed of heptamers of protective antigen (PA), which drives the endocytosis and translocation into host cells of the lethal factor (LF), a mitogen-activated protein kinase kinase protease. Here we investigated the consequences of injection of an endothelium-permeabilizing factor using LT as a “molecular syringe.” To this end, we generated the chimeric factor LE, corresponding to the PA-binding domain of LF (LF
1-254
) fused to EDIN exoenzyme. EDIN ADP ribosylates RhoA, leading to actin cable disruption and formation of transcellular tunnels in endothelial cells. We report that systemic injection of LET (LE plus PA) triggers a PA-dependent increase in the pulmonary endothelium permeability. We also report that native LT induces a progressive loss of endothelium barrier function. We established that there is a direct correlation between the extent of endothelium permeability induced by LT and the cytotoxic activity of LT. This suggests new ways to design therapeutic drugs against anthrax directed toward vascular permeability.
Decreased Expression of Colonic Slc26a3 and Carbonic Anhydrase IV as a Cause of Fatal Infectious Diarrhea in Mice
Authors: Borenshtein Diana , Schlieper Katherine A. , Rickman Barry H. , Chapman Jeannie M. , Schweinfest Clifford W. , Fox James G. , Schauer David B.
Published: June 2009

Citrobacter rodentiumcauses epithelial hyperplasia and colitis and is used as a model for enteropathogenic and enterohemorrhagicEscherichia coliinfections. Little or no mortality develops in most inbred strains of mice, but C3H and FVB/N mice exhibit fatal outcomes of infection. Here we test the hypothesis that decreased intestinal transport activity duringC. rodentiuminfection results in fatality in C3H/HeOu and FVB/N mice. Susceptible strains were compared to resistant C57BL/6 mice and to inbred strains SWR and SJL of Swiss origin, which have not been previously characterized for outcomes ofC. rodentiuminfection. Mortality in susceptible strains C3H/HeOu and FVB/N was associated with significant fluid loss in feces, a remarkable downregulation of Slc26a3 and carbonic anhydrase IV (CAIV) message and protein expression, retention of chloride in stool, and hypochloremia, suggesting defects in intestinal chloride absorption. SWR, SJL, and C57BL/6 mice were resistant and survived the infection. Fluid therapy fully prevented mortality in C3H/HeOu and FVB/N mice without affecting clinical disease. Common pathogenic mechanisms, such as decreased levels of expression of Slc26a3 and CAIV, affect intestinal ion transport inC. rodentium-infected FVB and C3H mice, resulting in profound electrolyte loss, dehydration, and mortality. Intestinal chloride absorption pathways are likely a potential target for the treatment of infectious diarrhea.
Outer Membrane Protein Expression Profile in
Helicobacter pylori
Clinical Isolates
Authors: Odenbreit Stefan , Swoboda Kirstin , Barwig Iris , Ruhl Stefan , Borén Thomas , Koletzko Sibylle , Haas Rainer
Published: June 2009

The gram-negative gastric pathogen
Helicobacter pylori
is equipped with an extraordinarily large set of outer membrane proteins (OMPs), whose role in the infection process is not well understood. The Hop (
Helicobacter o
uter membrane
p
orins) and Hor (
Ho
p-
r
elated proteins) groups constitute a large paralogous family consisting of 33 members. The OMPs AlpA, AlpB, BabA, SabA, and HopZ have been identified as adhesins or adherence-associated proteins. To better understand the relevance of these and other OMPs during infection, we analyzed the expression of eight different
omp
genes (
alpA
,
alpB
,
babA
,
babB
,
babC
,
sabA
,
hopM
, and
oipA
) in a set of 200 patient isolates, mostly from symptomatic children or young adults. Virtually all clinical isolates produced the AlpA and AlpB proteins, supporting their essential function. All other OMPs were produced at extremely variable rates, ranging from 35% to 73%, indicating a function in close adaptation to the individual host or gastric niche. In 11% of the isolates, BabA was produced, and SabA was produced in 5% of the isolates, but the strains failed to bind their cognate substrates. Interleukin-8 (IL-8) expression in gastric cells was strictly dependent on the presence of the
cag
pathogenicity island, whereas the presence of OipA clearly enhanced IL-8 production. The presence of the translocated effector protein CagA correlated well with BabA and OipA production. In conclusion, we found unexpectedly diverse
omp
expression profiles in individual
H. pylori
strains and hypothesize that this reflects the selective pressure for adhesion, which may differ across different hosts as well as within an individual over time.
TheAcinetobacter baylyi hfqGene Encodes a Large Protein with an Unusual C Terminus
Authors: Schilling Dominik , Gerischer Ulrike
Published: June 2009

In gammaproteobacteria the Hfq protein shows a great variation in size, especially in its C-terminal part. Extremely large Hfq proteins consisting of almost 200 amino acid residues and more are found within the gammaproteobacterial familyMoraxellaceae. The difference in size compared to other Hfq proteins is due to a glycine-rich domain near the C-terminal end of the protein.Acinetobacter baylyi, a nonpathogenic soil bacterium and member of theMoraxellaceaeencodes a large 174-amino-acid Hfq homologue containing the unique and repetitive amino acid pattern GGGFGGQ within the glycine-rich domain. Despite the presence of the C-terminal extension,A. baylyiHfq complemented anEscherichia coli hfqmutant in vivo. By using polyclonal anti-Hfq antibodies, we detected the largeA. baylyiHfq that corresponds to its annotated size indicating the expression and stability of the full protein. Deletion of the completeA. baylyi hfqopen reading frame resulted in severe reduction of growth. In addition, a deletion or overexpression of Hfq was accompanied by the loss of cell chain assembly. The glycine-rich domain was not responsible for growth and cell phenotypes.hfqgene localization inA. baylyiis strictly conserved within themutL-miaA-hfqoperon, and we show thathfqexpression starts within the precedingmiaAgene or further upstream.
An efficient synthesis of 6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-pyrrolizine, a key intermediate in the licofelone synthesis
Authors: Rádl Stanislav , Stach Jan , Černý Josef , Klecán Ondřej
Published: June 2009

An efficient synthesis of 6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-pyrrolizine, a key intermediate for the synthesis of licofelone, an anti-inflammatory drug currently undergoing evaluation of the phase-III clinical studies, is described. The method is based on a novel synthesis of unstable 5-benzyl-3,3-dimethyl-3,4-dihydro-2H-pyrrole, which is then treated with 2-bromo-1-(4-chlorophenyl)ethan-1-one. 2,2-Dimethyl-5-phenylpent-4-ynal with benzylamines provides the corresponding Schiff bases. Migration of the C=N double bond in these N-(2,2-dimethyl-5-phenylpent-4-yn-1-ylidene)benzylamines into conjugation with the aromatic ring using various base/solvent systems was studied. Acid hydrolysis of the formed Schiff bases then provided 2,2-dimethyl-5-phenylpent-4-yn-1-amine and 2,2-dimethyl-5-phenylpenta-3,4-dien-1-amine; their ratio was influenced mainly by the reaction conditions. Cyclization of these amines using Ag or Au catalysts then led to 5-benzyl-3,3-dimethyl-3,4-dihydro-2H-pyrrole.
Exposure–response relationship between lung cancer and polycyclic aromatic hydrocarbons (PAHs)
Authors: Armstrong B G , Gibbs G
Published: June 2009

Objectives:

To estimate the exposure–response function associating polycyclic aromatic hydrocarbon (PAH) exposure and lung cancer, with consideration of smoking.

Methods:

Mortality, occupational exposure and smoking histories were ascertained for a cohort of 16 431 persons (15 703 men and 728 women) who had worked in one of four aluminium smelters in Quebec from 1950 to 1999. A variety of exposure–response functions were fitted to the cohort data using generalised relative risk models.

Results:

In 677 lung cancer cases there was a clear trend of increasing risk with increasing cumulative exposure to PAH measured as benzo(a)pyrene (BaP). A linear model predicted a relative risk of 1.35 (95% CI 1.22 to 1.51) at 100 μg/m−3BaP years, but there was a significant departure from linearity in the direction of decreasing slope with increasing exposures. Among the models tried, the best fitting were a two-knot cubic spline and a power curve (RR = (1+bx)p), the latter predicting a relative risk of 2.68 at 100 μg/m−3BaP years. Additive models and multiplicative models for combining risks from occupational PAH and smoking fitted almost equally well, with a slight advantage to the additive.

Conclusion:

Despite the large cohort with long follow-up, the shape of the exposure–response function and the mode of combination of risks due to occupational PAH and smoking remains uncertain. If a linear exposure–response function is assumed, the estimated slope is broadly in line with the estimate from a previous follow-up of the same cohort, and somewhat higher than the average found in a recent meta-analysis of lung cancer studies.
Some Load Tests on Drilled Cast -in-Place Concrete Caissons
Authors: Matich M J , Kozicki P
Published: June 2009

A number of load tests carried out on drilled cast-in-place concrete caissons are described, along with some pertinent geo-technical properties of the glacial till overburden and shale bedrock. Both the till and the shale deteriorated rapidly under the combined effects of exposure by excavation and action of groundwater seepage. The unusual features of the project were that straight shaft caissons were used in such conditions, and that they were designed for side shear only.
Yielding and Flow of Sand in Triaxial Compression: Parts II and III
Authors: Poorooshasb H B , Holubec I , Sherbourne A N
Published: June 2009

In Part I,* based on experimental evidence, the existence of a potential function to define the gradient of the plastic strain increment vector was proved. The study is continued in Part II by defining the term yielding, a discussion of the form of the yield loci and a presentation of the “flow rule.”
Heaving Pressure in Soils During Unidirectional Freezing
Authors: Penner E
Published: June 2009

Ice lenses in natural soil are normally formed in a thermal gradient regime, and this permits the development of an undulating frost line if a range of pore sizes exists. Evidence is presented that suggests that the undulating nature of the ice-water interface permits the ice lens to form over the smaller pores of the system. Although fractions with only a limited particle size range have been studied, the results support the use of particle size as a valid basis for assessing frost susceptibility.
TRAVELING WAVE SOLUTIONS OF SEVENTH-ORDER GENERALIZED KdV EQUATIONS BY VARIATIONAL ITERATION METHOD USING ADOMIAN'S POLYNOMIALS
Authors: MOHYUD-DIN SYED TAUSEEF , NOOR MUHAMMAD ASLAM , NOOR KHALIDA INAYAT
Published: June 2009

In this paper, we apply the variational iteration method using Adomian's polynomials (VIMAP) to investigate propagating traveling solitary wave solutions of seventh-order generalized KdV (SOG-KdV) equations, which play a very important role in mathematical physics, engineering, and applied sciences. The suggested algorithm is quite efficient and is practically well suited for use in these problems. The proposed iterative scheme finds the solution without any discretization, perturbation, linearization, or restrictive assumptions and is formulated by the elegant coupling of variational iteration method and Adomian's polynomials.
THE QUANTIZATION OF A MESOSCOPIC TRANSMISSION LINE WITH THE DISCRETENESS OF ELECTRIC CHARGE
Authors: JI YING-HUA
Published: June 2009

The quantization of a mesoscopic transmission line in accordance with the discreteness of electric charges is proposed under the Born–Von-Karmann periodic boundary condition. The finite-difference Schrödinger equation of the circuit has been obtained in charge representation, and the Schrödinger equation is transformed into Mathieu equation in current representation. The wavefunction and energy spectrum can be solved by adopting the canonical transformation and unitary transformation method. Our results indicate that in the transmission line, quantum fluctuation of currents, which also have distributed properties, are related to both the circuit parameters and the positions and the mode of signals.
SCHEME FOR IMPLEMENTING ASSISTED CLONING OF AN UNKNOWN TWO-PARTICLE ENTANGLED STATE VIA CLUSTER STATE
Authors: MA PENG-CHENG , ZHAN YOU-BANG , ZHANG LING-LING
Published: June 2009

We present a scheme that can realize quantum cloning of an unknown two-particle entangled state and its orthogonal-complement state with assistance offered by a state preparer, via entangled cluster state as quantum channel. The first stage of the scheme requires usual teleportation via a maximally four-particle entangled cluster state as quantum channel. In the second stage of the scheme, with the assistance (through a two-particle projective measurement) of the preparer, the perfect copies and complement copies of an unknown two-particle entangled state can be obtained. We also put forward a scheme for the teleportation by using nonmaximally entangled quantum channel. The clones and complement clones of unknown state can be produced with certain probability in the latter scheme.
ON THE FERROMAGNETIC PHASE TRANSITION IN NANO ZnO:Mn BY ELECTRON PARAMAGNETIC RESONANCE
Authors: LAKSHMI P. VINOTHA BOORANA , RANGANATHAN B. , RAJKUMAR N. , RAMACHANDRAN K.
Published: June 2009

Nanocrystalline samples of Zn 1-x Mn x O (x = 0.0, 0.01, 0.02, 0.1 and 0.2) were synthesized by solid state reaction and characterized by X-ray diffraction and electron paramagnetic resonance (EPR) at room temperature. EPR studies showed an evidence of ferromagnetism above room temperature.
EFFECT OF BIAS VOLTAGE ON THE FATIGUE LIFE OF MARTENSITIC STAINLESS STEEL WITH TiN FILM COATED USING ARC ION PLATING METHOD
Authors: FUKUI SATOSHI , YONEKURA DAISUKE , MURAKAMI RI-ICHI
Published: June 2009

To investigate the effect of TiN coating on the fatigue strength of high-strength steel, four-point bending fatigue tests were carried out for martensitic stainless steel with TiN film coated using arc ion plating (AIP) method. A 2-μm-thick TiN film was deposited onto the substrate surface under bias voltage of four kinds: VB = 0, -60, -160 and -260 V. For VB = 0, -160 V and -260 V, the fatigue limit increased. The highest fatigue limit of σ max = 900 MPa was obtained for VB = -160 V. But some samples for VB = -260 V showed the decrease of fatigue limit due to film delamination during the fatigue test. For VB = -60 V, the fatigue limit was unchanged by coating. As a result of a coating property analysis, the following conclusions were obtained. Fatigue crack propagation was almost independent of the bias voltage. Fatigue crack initiated from the subsurface in the substrate and the crack initiation behavior depended on the film property of the adhesion, residual stress, elastic modulus, and the film's hardness depended on the bias voltage especially for low fatigue stress level.
LINEAR ELECTRO-OPTICAL EFFECT ON DPS-DNG LAYERED STRUCTURE
Authors: SHAHBAZIAN J. H. , KARAKASHIAN A. S.
Published: June 2009

In this letter, the influence of an applied electric field on a periodic structure composed of alternating layers of DPS (Double Positive index of refraction) and DNG (Double Negative index of refraction) has been studied. We present theoretical and numerical results of our investigation of the reflection spectra of the visible waves on this structure in the presence of an electric field which will cause redistribution of charges and will modify the index of refraction of materials and enable us to control the phase and/or intensity of propagating electromagnetic waves.
POLARIZED ATOMIC FERMI GASES
Authors: LI WENHUI , PARTRIDGE G. B. , LIAO Y. A. , HULET R. G.
Published: June 2009

We review recent experimental studies on polarized atomic Fermi gases. The particular focus is on our own experiments of strongly interacting two-component Fermi gases with imbalanced populations. The real-space density distributions reveal a superfluid/normal phase separation at very low temperatures, accompanied by deformation of evenly paired core; at higher temperatures, a partially polarized phase is observed.
GLOBAL WEAKLY DISCONTINUOUS SOLUTIONS TO THE MIXED INITIAL–BOUNDARY VALUE PROBLEM FOR QUASILINEAR HYPERBOLIC SYSTEMS
Authors: SHAO ZHI-QIANG
Published: June 2009

In this paper, we consider the mixed initial–boundary value problem for first-order quasilinear hyperbolic systems with general nonlinear boundary conditions in the half space {(t, x) | t ≥ 0, x ≥ 0}. Based on the fundamental local existence results and global-in-time a priori estimates, we prove the global existence of a unique weakly discontinuous solution u = u(t, x) with small and decaying initial data, provided that each characteristic with positive velocity is weakly linearly degenerate. Some applications to quasilinear hyperbolic systems arising in physics and other disciplines, particularly to the system describing the motion of the relativistic closed string in the Minkowski space R1+n, are also given.
MATHEMATICAL FRAMEWORK OF THE WELL PRODUCTIVITY INDEX FOR FAST FORCHHEIMER (NON-DARCY) FLOWS IN POROUS MEDIA
Authors: AULISA EUGENIO , IBRAGIMOV AKIF , VALKO PETER , WALTON JAY
Published: June 2009

Motivated by the reservoir engineering concept of the well Productivity Index, we introduced and analyzed a functional, denoted as "diffusive capacity", for the solution of the initial-boundary value problem (IBVP) for a linear parabolic equation.21 This IBVP described laminar (linear) Darcy flow in porous media; the considered boundary conditions corresponded to different regimes of the well production. The diffusive capacities were then computed as steady state invariants of the solutions to the corresponding time-dependent boundary value problem.

Here similar features for fast or turbulent nonlinear flows subjected to the Forchheimer equations are analyzed. It is shown that under some hydrodynamic and thermodynamic constraints, there exists a so-called pseudo steady state regime for the Forchheimer flows in porous media. In other words, under some assumptions there exists a steady state invariant over a certain class of solutions to the transient IBVP modeling the Forchheimer flow for slightly compressible fluid. This invariant is the diffusive capacity, which serves as the mathematical representation of the so-called well Productivity Index. The obtained results enable computation of the well Productivity Index by resolving a single steady state boundary value problem for a second-order quasilinear elliptic equation. Analytical and numerical studies highlight some new relations for the well Productivity Index in linear and nonlinear cases. The obtained analytical formulas can be potentially used for the numerical well block model as an analog of Piecemann.
ON A MODEL FOR THE PROPAGATION OF ISOTOPIC DISEQUILIBRIUM BY DIFFUSION
Authors: COMPARINI E. , DAL PASSO R. , PESCATORE C. , UGHI M.
Published: June 2009

We consider a model for the distribution of radionuclides in the ground water around a deep repository for used nuclear fuel, based on the assumption that different isotopes of the same chemical element A contribute jointly to the chemical potential of A. In this hypothesis, the total flux Ji of a particular isotope Ai of an element A has two components, one due to the interaction of Ai with the solvent molecules B, the other with the kin isotopes. We study some qualitative properties of the solution in the physically relevant assumption that the first of these components is negligible. In this assumption the problem reduces to a parabolic equation for the total concentration of the element A, possibly coupled with hyperbolic equations for the concentrations of the single isotopes.