bluuebunny/crossref_metadata_2025 huggingface dataset:
Hepatic expression of gamma‐glutamyltranspeptidase in the human liver of patients with alcoholic liver disease
Authors: Irie Makoto , Suzuki Norihisa , Sohda Tetsuro , Anan Akira , Iwata Kaoru , Takeyama Yasuaki , Watanabe Hiroshi , Fischer Peter , Scherberich Jürgen E. , Sakisaka Shotaro
Published: June 2007

Background:  Gamma‐glutamyltranspeptidase (GGT) has been recognized as an enzyme that converts glutathione into cysteine, and it is localized predominantly within the liver. Serum GGT is clinically recognized as the most useful marker for diagnosis of alcoholic liver disease (ALD).

Methods:  GGT localization within the liver was examined immunohistochemically using an anti‐GGT antibody and was visualized by confocal laser scanning microscopy in ALD and normal livers. Double immunostaining for GGT and dipeptidylpeptidase‐IV (DPP‐IV) was carried out to evaluate GGT localization in greater detail.

Results:  Expression of GGT protein and mRNA was studied with immunoblot analysis and in situ hybridization, respectively. Immunohistochemically, the expression of GGT in the normal liver was faintly demonstrated in the bile canaliculi of hepatocytes and in biliary epithelial cells. In ALD livers, GGT was clearly demonstrated at the same sites. Double immunostaining demonstrated that GGT and DPP‐IV were colocalized in hepatocytes in the ALD liver. In situ hybridization clearly demonstrated GGT‐mRNA within the cytoplasm of hepatocytes and biliary epithelial cells. Immunoblot analysis revealed that GGT protein expression was increased in the ALD livers compared with that seen in the normal livers.

Conclusion:  These findings indicate that GGT in control and alcoholic livers is synthesized in hepatocytes and biliary epithelial cells, and is localized within the bile canalicular membrane and the luminal membrane in those cells, respectively. In conclusion, GGT synthesis and protein expression are increased in ALD livers, leading to the elevation of serum levels of GGT that are commonly noted in patients with the disease.
The Coprophilous Mushroom
Coprinus radians
Secretes a Haloperoxidase That Catalyzes Aromatic Peroxygenation
Authors: Anh Dau Hung , Ullrich René , Benndorf Dirk , Svatoś Aleś , Muck Alexander , Hofrichter Martin
Published: June 2007

Coprophilous and litter-decomposing species (26 strains) of the genus
Coprinus
were screened for peroxidase activities by using selective agar plate tests and complex media based on soybean meal. Two species,
Coprinus radians
and
C. verticillatus
, were found to produce peroxidases, which oxidized aryl alcohols to the corresponding aldehydes at pH 7 (a reaction that is typical for heme-thiolate haloperoxidases). The peroxidase of
Coprinus radians
was purified to homogeneity and characterized. Three fractions of the enzyme, CrP I, CrP II, and CrP III, with molecular masses of 43 to 45 kDa as well as isoelectric points between 3.8 and 4.2, were identified after purification by anion-exchange and size exclusion chromatography. The optimum pH of the major fraction (CrP II) for the oxidation of aryl alcohols was around 7, and an H
2
O
2
concentration of 0.7 mM was most suitable regarding enzyme activity and stability. The apparent
K

m

values for ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)], 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, and H
2
O
2
were 49, 342, 635, 88, and 1,201 μM, respectively. The N terminus of CrP II showed 29% and 19% sequence identity to
Agrocybe aegerita
peroxidase (AaP) and chloroperoxidase, respectively. The UV-visible spectrum of CrP II was highly similar to that of resting-state cytochrome P450 enzymes, with the Soret band at 422 nm and additional maxima at 359, 542, and 571 nm. The reduced carbon monoxide complex showed an absorption maximum at 446 nm, which is characteristic of heme-thiolate proteins. CrP brominated phenol to 2- and 4-bromophenols and selectively hydroxylated naphthalene to 1-naphthol. Hence, after AaP, CrP is the second extracellular haloperoxidase-peroxygenase described so far. The ability to extracellularly hydroxylate aromatic compounds seems to be the key catalytic property of CrP and may be of general significance for the biotransformation of poorly available aromatic substances, such as lignin, humus, and organopollutants in soil litter and dung environments. Furthermore, aromatic peroxygenation is a promising target of biotechnological studies.
The Tomato Rhizosphere, an Environment Rich in Nitrogen-Fixing
Burkholderia
Species with Capabilities of Interest for Agriculture and Bioremediation
Authors: Caballero-Mellado Jesús , Onofre-Lemus Janette , Estrada-de los Santos Paulina , Martínez-Aguilar Lourdes
Published: June 2007

Burkholderia
strains are promising candidates for biotechnological applications. Unfortunately, most of these strains belong to species of the
Burkholderia cepacia
complex (Bcc) involved in human infections, hampering potential applications. Novel diazotrophic
Burkholderia
species, phylogenetically distant from the Bcc species, have been discovered recently, but their environmental distribution and relevant features for agro-biotechnological applications are little known. In this work, the occurrence of N
2
-fixing
Burkholderia
species in the rhizospheres and rhizoplanes of tomato plants field grown in Mexico was assessed. The results revealed a high level of diversity of diazotrophic
Burkholderia
species, including
B. unamae
,
B. xenovorans
,
B. tropica
, and two other unknown species, one of them phylogenetically closely related to
B. kururiensis
. These N
2
-fixing
Burkholderia
species exhibited activities involved in bioremediation, plant growth promotion, or biological control in vitro. Remarkably,
B. unamae
and
B. kururiensis
grew with aromatic compounds (phenol and benzene) as carbon sources, and the presence of aromatic oxygenase genes was confirmed in both species. The rhizospheric and endophyte nature of
B. unamae
and its ability to degrade aromatic compounds suggest that it could be used in rhizoremediation and for improvement of phytoremediation.
B. kururiensis
and other
Burkholderia
sp. strains grew with toluene.
B. unamae
and
B. xenovorans
exhibited ACC (1-aminocyclopropane-1-carboxylic acid) deaminase activity, and the occurrence of
acdS
genes encoding ACC deaminase was confirmed. Mineral phosphate solubilization through organic acid production appears to be the mechanism used by most diazotrophic
Burkholderia
species, but in
B. tropica
, there presumably exists an additional unknown mechanism. Most of the diazotrophic
Burkholderia
species produced hydroxamate-type siderophores. Certainly, the N
2
-fixing
Burkholderia
species associated with plants have great potential for agro-biotechnological applications.
Development of Innovative Pediocin PA-1 by DNA Shuffling among Class IIa Bacteriocins
Authors: Tominaga Tatsuya , Hatakeyama Yoshinori
Published: June 2007

Pediocin PA-1 is a member of the class IIa bacteriocins, which show antimicrobial effects against lactic acid bacteria. To develop an improved version of pediocin PA-1, reciprocal chimeras between pediocin PA-1 and enterocin A, another class IIa bacteriocin, were constructed. Chimera EP, which consisted of the C-terminal half of pediocin PA-1 fused to the N-terminal half of enterocin A, showed increased activity against a strain of
Leuconostoc lactis
isolated from a sour-spoiled dairy product. To develop an even more effective version of this chimera, a DNA-shuffling library was constructed, wherein four specific regions within the N-terminal half of pediocin PA-1 were shuffled with the corresponding sequences from 10 other class IIa bacteriocins. Activity screening indicated that 63 out of 280 shuffled mutants had antimicrobial activity. A colony overlay activity assay showed that one of the mutants (designated B1) produced a >7.8-mm growth inhibition circle on
L. lactis
, whereas the parent pediocin PA-1 did not produce any circle. Furthermore, the active shuffled mutants showed increased activity against various species of
Lactobacillus
,
Pediococcus
, and
Carnobacterium
. Sequence analysis revealed that the active mutants had novel N-terminal sequences; in active mutant B1, for example, the parental pediocin PA-1 sequence (KYYGNGV
T
C
GKHS
C) was changed to
T
KYYGNGV
S
C
TKSG
C. These new and improved DNA-shuffled bacteriocins could prove useful as food additives for inhibiting sour spoilage of dairy products.
Characterization of a
Bifidobacterium longum
BORI Dipeptidase Belonging to the U34 Family
Authors: Seo Jeong Min , Ji Geun Eog , Cho Sang Hee , Park Myung Soo , Lee Hyong Joo
Published: June 2007

A dipeptidase was purified from a cell extract of
Bifidobacterium longum
BORI by ammonium sulfate precipitation and chromatography on DEAE-cellulose and Q-Sepharose columns. The purified dipeptidase had a molecular mass of about 49 kDa and was optimally active at pH 8.0 and 50°C. The enzyme was a strict dipeptidase, being capable of hydrolyzing a range of dipeptides but not tri- and tetrapeptides,
p
-nitroanilide derivatives of amino acids, or N- or C-terminus-blocked dipeptides. A search of the amino acid sequence of an internal tryptic fragment against protein sequences deduced from the total genome sequence of
B. longum
NCC2705 revealed that it was identical to an internal sequence of the dipeptidase gene (
pepD
), which comprised 1,602 nucleotides encoding 533 amino acids with a molecular mass of 60 kDa, and thereby differed considerably from the 49-kDa mass of the purified dipeptidase. To understand this discrepancy,
pepD
was cloned into an
Escherichia coli
expression vector (pBAD-TOPO derivative) to generate the recombinant plasmids pBAD-pepD and pBAD-pepD-His (note that His in the plasmid designation stands for a polyhistidine coding region). Both plasmids were successfully expressed in
E. coli
, and the recombinant protein PepD-His was purified using nickel-chelating affinity chromatography and reconfirmed by internal amino acid sequencing. The PepD sequence was highly homologous to those of the U34 family of peptidases, suggesting that the
B. longum
BORI dipeptidase is a type of cysteine-type N-terminal nucleophile hydrolase and has a β-hairpin motif similar to that of penicillin V acylase, which is activated by autoproteolytic processing.
Effects of Imposed Salinity Gradients on Dissimilatory Arsenate Reduction, Sulfate Reduction, and Other Microbial Processes in Sediments from Two California Soda Lakes
Authors: Kulp T. R. , Han S. , Saltikov C. W. , Lanoil B. D. , Zargar K. , Oremland R. S.
Published: June 2007

Salinity effects on microbial community structure and on potential rates of arsenate reduction, arsenite oxidation, sulfate reduction, denitrification, and methanogenesis were examined in sediment slurries from two California soda lakes. We conducted experiments with Mono Lake and Searles Lake sediments over a wide range of salt concentrations (25 to 346 g liter
−1
). With the exception of sulfate reduction, rates of all processes demonstrated an inverse relationship to total salinity. However, each of these processes persisted at low but detectable rates at salt saturation. Denaturing gradient gel electrophoresis analysis of partial 16S rRNA genes amplified from As(V) reduction slurries revealed that distinct microbial populations grew at low (25 to 50 g liter
−1
), intermediate (100 to 200 g liter
−1
), and high (>300 g liter
−1
) salinity. At intermediate and high salinities, a close relative of a cultivated As-respiring halophile was present. These results suggest that organisms adapted to more dilute conditions can remain viable at high salinity and rapidly repopulate the lake during periods of rising lake level. In contrast to As reduction, sulfate reduction in Mono Lake slurries was undetectable at salt saturation. Furthermore, sulfate reduction was excluded from Searles Lake sediments at any salinity despite the presence of abundant sulfate. Sulfate reduction occurred in Searles Lake sediment slurries only following inoculation with Mono Lake sediment, indicating the absence of sulfate-reducing flora. Experiments with borate-amended Mono Lake slurries suggest that the notably high (0.46 molal) concentration of borate in the Searles Lake brine was responsible for the exclusion of sulfate reducers from that ecosystem.
Molecular Detection and Ecological Significance of the Cyanobacterial Genera
Geitlerinema
and
Leptolyngbya
in Black Band Disease of Corals
Authors: Myers Jamie L. , Sekar Raju , Richardson Laurie L.
Published: June 2007

Black band disease (BBD) is a pathogenic, sulfide-rich microbial mat dominated by filamentous cyanobacteria that infect corals worldwide. We isolated cyanobacteria from BBD into culture, confirmed their presence in the BBD community by using denaturing gradient gel electrophoresis (DGGE), and demonstrated their ecological significance in terms of physiological sulfide tolerance and photosynthesis-versus-irradiance values. Twenty-nine BBD samples were collected from nine host coral species, four of which have not previously been investigated, from reefs of the Florida Keys, the Bahamas, St. Croix, and the Philippines. From these samples, seven cyanobacteria were isolated into culture. Cloning and sequencing of the 16S rRNA gene using universal primers indicated that four isolates were related to the genus
Geitlerinema
and three to the genus
Leptolyngbya
. DGGE results, obtained using
Cyanobacteria
-specific 16S rRNA primers, revealed that the most common BBD cyanobacterial sequence, detected in 26 BBD field samples, was related to that of an
Oscillatoria
sp. The next most common sequence, 99% similar to that of the
Geitlerinema
BBD isolate, was present in three samples. One
Leptolyngbya
- and one
Phormidium
-related sequence were also found. Laboratory experiments using isolates of BBD
Geitlerinema
and
Leptolyngbya
revealed that they could carry out sulfide-resistant oxygenic photosynthesis, a relatively rare characteristic among cyanobacteria, and that they are adapted to the sulfide-rich, low-light BBD environment. The presence of the cyanotoxin microcystin in these cultures and in BBD suggests a role in BBD pathogenicity. Our results confirm the presence of
Geitlerinema
in the BBD microbial community and its ecological significance, which have been challenged, and provide evidence of a second ecologically significant BBD cyanobacterium,
Leptolyngbya
.
Rhizobacterial Volatiles Affect the Growth of Fungi and
Arabidopsis thaliana
Authors: Vespermann Anja , Kai Marco , Piechulla Birgit
Published: June 2007

Volatiles of
Stenotrophomonas
,
Serratia
, and
Bacillus
species inhibited mycelial growth of many fungi and
Arabidopsis thaliana
(40 to 98%), and volatiles of
Pseudomonas
species and
Burkholderia cepacia
retarded the growth to lesser extents.
Aspergillus niger
and
Fusarium
species were resistant, and
B. cepacia
and
Staphylococcus epidermidis
promoted the growth of
Rhizoctonia solani
and
A. thaliana
. Bacterial volatiles provide a new source of compounds with antibiotic and growth-promoting features.
Glutathione Protects
Lactococcus lactis
against Acid Stress
Authors: Zhang Juan , Fu Rui-Yan , Hugenholtz Jeroen , Li Yin , Chen Jian
Published: June 2007

Previously we showed that glutathione (GSH) can protect
Lactococcus lactis
against oxidative stress (Y. Li et al., Appl. Environ. Microbiol. 69:5739-5745, 2003). In the present study, we show that the GSH imported by
L. lactis
subsp.
cremoris
SK11 or produced by engineered
L. lactis
subsp.
cremoris
NZ9000 can protect both strains against a long-term mild acid challenge (pH 4.0) and a short-term severe acid challenge (pH 2.5). This shows for the first time that GSH can protect a gram-positive bacterium against acid stress. During acid challenge, strain SK11 containing imported GSH and strain NZ9000 containing self-produced GSH exhibited significantly higher intracellular pHs than the control. Furthermore, strain SK11 containing imported GSH had a significantly higher activity of glyceraldehyde-3-phosphate dehydrogenase than the control. These results suggest that the acid stress resistance of starter culture can be improved by selecting
L. lactis
strains capable of producing or importing GSH.
Intracellular Siderophores Are Essential for Ascomycete Sexual Development in Heterothallic
Cochliobolus heterostrophus
and Homothallic
Gibberella zeae
Authors: Oide Shinichi , Krasnoff Stuart B. , Gibson Donna M. , Turgeon B. Gillian
Published: June 2007

Connections between fungal development and secondary metabolism have been reported previously, but as yet, no comprehensive analysis of a family of secondary metabolites and their possible role in fungal development has been reported. In the present study, mutant strains of the heterothallic ascomycete
Cochliobolus heterostrophus
, each lacking one of 12 genes (
NPS1
to
NPS12
) encoding a nonribosomal peptide synthetase (NRPS), were examined for a role in sexual development. One type of strain (Δ
nps2
) was defective in ascus/ascospore development in homozygous Δ
nps2
crosses. Homozygous crosses of the remaining 11 Δ
nps
strains showed wild-type (WT) fertility. Phylogenetic, expression, and biochemical analyses demonstrated that the NRPS encoded by
NPS2
is responsible for the biosynthesis of ferricrocin, the intracellular siderophore of
C. heterostrophus
. Functional conservation of
NPS2
in both heterothallic
C. heterostrophus
and the unrelated homothallic ascomycete
Gibberella zeae
was demonstrated.
G. zeae
Δ
nps2
strains are concomitantly defective in intracellular siderophore (ferricrocin) biosynthesis and sexual development. Exogenous application of iron partially restored fertility to
C. heterostrophus
and
G. zeae
Δ
nps2
strains, demonstrating that abnormal sexual development of Δ
nps2
strains is at least partly due to their iron deficiency. Exogenous application of the natural siderophore ferricrocin to
C. heterostrophus
and
G. zeae
Δ
nps2
strains restored WT fertility.
NPS1
, a
G. zeae NPS
gene that groups phylogenetically with
NPS2
, does not play a role in sexual development. Overall, these data demonstrate that iron and intracellular siderophores are essential for successful sexual development of the heterothallic ascomycete
C. heterostrophus
and the homothallic ascomycete
G. zeae
.
Gliotoxin Is a Virulence Factor of
Aspergillus fumigatus
:
gliP
Deletion Attenuates Virulence in Mice Immunosuppressed with Hydrocortisone
Authors: Sugui Janyce A. , Pardo Julian , Chang Yun C. , Zarember Kol A. , Nardone Glenn , Galvez Eva M. , Müllbacher Arno , Gallin John I. , Simon Markus M. , Kwon-Chung Kyung J.
Published: June 2007

Gliotoxin is an immunosuppressive mycotoxin long suspected to be a potential virulence factor of
Aspergillus fumigatus
. Recent studies using mutants lacking gliotoxin production, however, suggested that the mycotoxin is not important for pathogenesis of
A. fumigatus
in neutropenic mice resulting from treatment with cyclophosphomide and hydrocortisone. In this study, we report on the pathobiological role of gliotoxin in two different mouse strains, 129/Sv and BALB/c, that were immunosuppressed by hydrocortisone alone to avoid neutropenia. These strains of mice were infected using the isogenic set of a wild type strain (B-5233) and its mutant strain (
gliP
Δ) and the the
glip
reconstituted strain (
gliP
R
). The
gliP
gene encodes a nonribosomal peptide synthase that catalyzes the first step in gliotoxin biosynthesis. The
gliP
Δ strain was significantly less virulent than strain B-5233 or
gliP
R
in both mouse models. In vitro assays with culture filtrates (CFs) of B-5233,
gliP
Δ, and
gliP
R
strains showed the following: (i) deletion of
gliP
abrogated gliotoxin production, as determined by high-performance liquid chromatography analysis; (ii) unlike the CFs from strains B-5233 and
gliP
R
,
gliP
Δ CFs failed to induce proapoptotic processes in EL4 thymoma cells, as tested by Bak conformational change, mitochondrial-membrane potential disruption, superoxide production, caspase 3 activation, and phosphatidylserine translocation. Furthermore, superoxide production in human neutrophils was strongly inhibited by CFs from strain B-5233 and the
gliP
R
strain, but not the
gliP
Δ strain. Our study confirms that gliotoxin is an important virulence determinant of
A. fumigatus
and that the type of immunosuppression regimen used is important to reveal the pathogenic potential of gliotoxin.
MpkA-Dependent and -Independent Cell Wall Integrity Signaling in
Aspergillus nidulans
Authors: Fujioka Tomonori , Mizutani Osamu , Furukawa Kentaro , Sato Natsuko , Yoshimi Akira , Yamagata Youhei , Nakajima Tasuku , Abe Keietsu
Published: June 2007

Cell wall integrity signaling (CWIS) maintains cell wall biogenesis in fungi, but only a few transcription factors (TFs) and target genes downstream of the CWIS cascade in filamentous fungi are known. Because a mitogen-activated protein kinase (MpkA) is a key CWIS enzyme, the transcriptional regulation of
mpkA
and of cell wall-related genes (CWGs) is important in cell wall biogenesis. We cloned
Aspergillus nidulans mpkA
;
rlmA
, a TF gene orthologous to
Saccharomyces cerevisiae RLM1
that encodes Rlm1p, a major Mpk1p-dependent TF that regulates the transcription of
MPK1
besides that of CWGs; and An
swi4
and An
swi6
, homologous to
S. cerevisiae SWI4
and
SWI6
, encoding the Mpk1p-activating TF complex Swi4p-Swi6p, which regulates CWG transcription in a cell cycle-dependent manner.
A. nidulans rlmA
and
mpkA
cDNA functionally complemented
S. cerevisiae rlm1
Δ and
mpk1
Δ mutants, respectively, but An
swi4
and An
swi6
cDNA did not complement
swi4
Δ and
swi6
Δ mutants. We constructed
A. nidulans rlmA
, An
swi4
and An
swi6
, and
mpkA
disruptants (
rlmA
Δ, An
swi4
Δ An
swi6
Δ, and
mpkA
Δ strains) and analyzed
mpkA
and CWG transcripts after treatment with a β-1,3-glucan synthase inhibitor (micafungin) that could activate MpkA via CWIS. Levels of
mpkA
transcripts in the mutants as well as those in the wild type were changed after micafungin treatment. The β-glucuronidase reporter gene controlled by the
mpkA
promoter was expressed in the wild type but not in the
mpkA
Δ strain. Thus,
mpkA
transcription seems to be autoregulated by CWIS via MpkA but not by RlmA or AnSwi4-AnSwi6. The transcription of most CWGs except α-1,3-glucan synthase genes (
agsA
and
agsB
) was independent of RlmA and AnSwi4-AnSwi6 and seemed to be regulated by non-MpkA signaling. The transcriptional regulation of
mpkA
and of CWGs via CWIS in
A. nidulans
differs significantly from that in
S. cerevisiae
.
The Plant Pathogen
Ralstonia solanacearum
Needs Aerotaxis for Normal Biofilm Formation and Interactions with Its Tomato Host
Authors: Yao Jian , Allen Caitilyn
Published: June 2007

Ralstonia solanacearum
is a soilborne pathogen that causes bacterial wilt of diverse plant species. To locate and infect host plant roots
R. solanacearum
needs taxis, the ability to move toward more favorable conditions. However, the specific signals that attract this pathogen were unknown. One candidate is aerotaxis, or energy taxis, which guides bacteria toward optimal intracellular energy levels. The
R. solanacearum
genome encodes two putative aerotaxis transducers. Cloned
R. solanacearum aer1
and
aer2
genes restored aerotaxis to an
Escherichia coli aer
mutant, demonstrating that both genes encode heterologously functional aerotaxis transducers. Site-directed mutants lacking
aer1
,
aer2
, or both
aer1
and
aer2
were significantly less able to move up an oxygen gradient than the wild-type parent strain; in fact, the aerotaxis of the
aer
mutants was indistinguishable from that of a completely nonmotile strain. Tomato plants inoculated with either the
aer2
or the
aer1/aer2
mutant had slightly delayed wilt disease development. Furthermore, the
aer1/aer2
double mutant was significantly impaired in the ability to rapidly localize on tomato roots compared to its wild-type parent. Unexpectedly, all nonaerotactic mutants formed thicker biofilms on abiotic surfaces than the wild type. These results indicate that energy taxis contributes significantly to the ability of
R. solanacearum
to locate and effectively interact with its host plants.
Interaction of the Gifsy-1 Xis Protein with the Gifsy-1
attP
Sequence
Authors: Flanigan Asa , Gardner Jeffrey F.
Published: June 2007

The Gifsy-1 phage integrates site specifically into the
Salmonella
chromosome via an integrase-mediated site-specific recombination mechanism. Initial genetic analysis suggests that Gifsy-1 integrase-mediated excision of the Gifsy-1 phage is influenced by proteins encoded by both the Gifsy-1 and the Gifsy-2 phages. Our studies show that the Gifsy-1 Xis protein regulates the directionality of integrase-mediated excision of the Gifsy-1 phage. Electrophoretic mobility shift assays, DNase I footprinting, dimethyl sulfate (DMS) interference assays, and DMS protection assays were used to identify a 31-base-pair sequence in the
attP
region to which the Gifsy-1 protein binds. The results suggest that this recombination directionality factor binds in vitro to three imperfect direct repeats, spaced 10 base pairs apart, in a sequential and cooperative manner in the absence of other phage-encoded proteins. Our studies suggest that, while the Gifsy-1 Xis does not require additional factors for specific and high-affinity binding, it may form a microfilament on DNA similar to that described for the phage lambda Xis protein.
Identification of the Major Protein Component of the Pneumococcal Eclipse Complex
Authors: Morrison Donald A. , Mortier-Barrière Isabelle , Attaiech Laetitia , Claverys Jean-Pierre
Published: June 2007

During genetic transformation of
Streptococcus pneumoniae
, single strands from native donor DNA enter competent cells, where they associate with an unidentified protein with a molecular mass of 15 to 20 kDa to form the eclipse complex. Using Western blotting, we identify the principal protein cofractionating with donor DNA in this complex as SsbB.
Identification of a Biosynthetic Gene Cluster and the Six Associated Lipopeptides Involved in Swarming Motility ofPseudomonas syringaepv. tomato DC3000
Authors: Berti Andrew D. , Greve Nathan J. , Christensen Quin H. , Thomas Michael G.
Published: June 2007

Pseudomonasspecies are known to be prolific producers of secondary metabolites that are synthesized wholly or in part by nonribosomal peptide synthetases. In an effort to identify additional nonribosomal peptides produced by these bacteria, a bioinformatics approach was used to “mine” the genome ofPseudomonas syringaepv. tomato DC3000 for the metabolic potential to biosynthesize previously unknown nonribosomal peptides. Herein we describe the identification of a nonribosomal peptide biosynthetic gene cluster that codes for proteins involved in the production of six structurally related linear lipopeptides. Structures for each of these lipopeptides were proposed based on amino acid analysis and mass spectrometry analyses. Mutations in this cluster resulted in the loss of swarming motility ofP. syringaepv. tomato DC3000 on medium containing a low percentage of agar. This phenotype is consistent with the loss of the ability to produce a lipopeptide that functions as a biosurfactant. This work gives additional evidence that mining the genomes of microorganisms followed by metabolite and phenotypic analyses leads to the identification of previously unknown secondary metabolites.
Alginate Production by
Pseudomonas putida
Creates a Hydrated Microenvironment and Contributes to Biofilm Architecture and Stress Tolerance under Water-Limiting Conditions
Authors: Chang Woo-Suk , van de Mortel Martijn , Nielsen Lindsey , Nino de Guzman Gabriela , Li Xiaohong , Halverson Larry J.
Published: June 2007

Biofilms exist in a variety of habitats that are routinely or periodically not saturated with water, and residents must integrate cues on water abundance (matric stress) or osmolarity (solute stress) into lifestyle strategies. Here we examine this hypothesis by assessing the extent to which alginate production by
Pseudomonas putida
strain mt-2 and by other fluorescent pseudomonads occurs in response to water limitations and how the presence of alginate in turn influences biofilm development and stress tolerance. Total exopolysaccharide (EPS) and alginate production increased with increasing matric, but not solute, stress severity, and alginate was a significant component, but not the major component, of EPS. Alginate influenced biofilm architecture, resulting in biofilms that were taller, covered less surface area, and had a thicker EPS layer at the air interface than those formed by an mt-2
algD
mutant under water-limiting conditions, properties that could contribute to less evaporative water loss. We examined this possibility and show that alginate reduces the extent of water loss from biofilm residents by using a biosensor to quantify the water potential of individual cells and by measuring the extent of dehydration-mediated changes in fatty acid composition following a matric or solute stress shock. Alginate deficiency decreased survival of desiccation not only by
P. putida
but also by
Pseudomonas aeruginosa
PAO1 and
Pseudomonas syringae
pv. syringae B728a. Our findings suggest that in response to water-limiting conditions, pseudomonads produce alginate, which influences biofilm development and EPS physiochemical properties. Collectively these responses may facilitate the maintenance of a hydrated microenvironment, protecting residents from desiccation stress and increasing survival.
Relationship between hip strength and frontal plane posture of the knee during a forward lunge
Authors: Thijs Youri , Van Tiggelen Damien , Willems Tine , De Clercq Dirk , Witvrouw Erik
Published: June 2007

Objective:

Excessive frontal plane knee movement during forward lunge movements might be associated with the occurrence of knee injuries in tennis. Here, we attempt to determine whether hip muscle strength is related to the frontal plane motion of the knee during a functional lunge movement.

Design:

A correlational study.

Participants:

A total of 84 healthy subjects (76 men, 8 women), with no history of knee or lower leg complaints.

Interventions:

Muscle strength of six hip muscle groups was measured using a handheld dynamometer. Subjects were videotaped during a forward lunge and peak knee valgus or varus angles were determined using a digital video analysis software program.

Main outcome measurements:

A correlation was examined between hip muscle strength and the amount of frontal plane movement of the knee during a forward lunge.

Results:

There were no significant differences in hip muscle strength between the valgus group and the varus group during the forward lunge movement. No significant correlation was found between the strength of the assessed hip muscles and the amount of movement into valgus/varus. In the varus group a moderate positive correlation was found between the External Rotation/Internal Rotation force ratio and the amount of knee varus during the forward lunge movement (r = 0.31, p = 0.03).

Conclusions:

The findings suggest that in healthy subjects hip muscle strength is not correlated to the amount of valgus/varus movement of the knee during a forward lunge. This suggests that other factors (eg, proprioception, core hip stability) might be more important in controlling knee movement during this tennis-specific movement.
Effect of a multistage ultra-endurance triathlon on body composition: World Challenge Deca Iron Triathlon 2006
Authors: Knechtle B , Salas Fraire O , Andonie J L , Kohler G
Published: June 2007

Objective:

To investigate the effect of a multistage ultra-endurance triathlon on body composition in ultra-triathletes.

Design:

Descriptive field study.

Setting:

The “World Challenge Deca Iron Triathlon 2006” in Monterrey, Mexico, in which every day for 10 consecutive days athletes had to perform the distance of one Ironman triathlon.

Subjects:

Eight male ultra-endurance athletes (mean (SD) age 40.6 (10.7) years, weight 76.4 (8.4) kg, height 175 (4) cm and body mass index (BMI) 24.7 (2.2) kg/m2).

Interventions:

None.

Main outcome measurements:

Determination of body mass, protein mass, body fat, per cent body fat, mineral mass, total body water, intracellular water, extracellular water and lean body mass with a direct segmental multifrequency bioelectrical impedance method before the race and after each stage in order to show changes in body composition.

Results:

A statistically significant decrease of body mass (−2.4 kg, p = 0.014), body fat (−5 kg, p = 0.0078) and per cent body fat (−6.4%, p = 0.0078) occurred at the end of the first day compared to values taken in the pre-race period. In contrast, at the same time, a statistically significant increase of protein mass (+0.7 kg, p = 0.035), mineral mass (+0.2 kg, p = 0.04), total body water (+1.8 litres, p = 0.042), intracellular water (+1.6 litres, p = 0.034) and lean body mass (+2.6 kg, p = 0.023) was shown. After the first day until the end of the challenge, body fat (−3 kg, p>0.05) and per cent body fat (−3.9%, p>0.05) showed a statistically significant decrease, whereas the other parameters showed no changes.

Conclusions:

Athletes taking part in a multistage ultra-endurance triathlon over 10 Ironman triathlon distances in 10 consecutive days lost 3 kg of body fat; skeletal muscle mass, mineral mass and body water were unchanged.
Duodenal follicular lymphomas share common characteristics with mucosa-associated lymphoid tissue lymphomas
Authors: Sato Y , Ichimura K , Tanaka T , Takata K , Morito T , Sato H , Kondo E , Yanai H , Ohara N , Oka T , Yoshino T
Published: June 2007

Background:

Follicular lymphomas occasionally arise in the extra-nodal organs and are frequently found in the duodenum. They are often localised tumours with multiple polyps around the ampulla of Vater.

Aims:

To examine a IgH/bcl-2 hybrid gene and VH gene to investigate the nature of the lymphoma cells and how they differ from nodal follicular lymphomas and MALT lymphomas.

Methods:

Of 40 patients reported previously, 35 with duodenal follicular lymphoma were studied in detail with respect to clinicopathological characteristics.

Results:

37/40 patients were in clinical stage I (n = 30) or stage II (n = 7). Clonal immunoglobulin gene rearrangement was detected in 53.3% of examined cases, and rearrangement of IgH/bcl-2 gene at the major break point was detected in 27% of cases. Three of 8 examined cases were VH4 (38%); 2 out of them were VH4-34. As VH4 deviation is one of the common characteristics of MALT lymphomas and 2/3 were identical, duodenal follicular lymphomas have a similar aetiology to MALT lymphomas. Clinical course was also similar to that of MALT lymphomas.

Conclusions:

Results suggest that duodenal follicular lymphomas have intermediate characteristics of MALT lymphomas and nodal follicular lymphomas.
Preservation of diastolic function in monocrotaline-induced right ventricular hypertrophy in rats
Authors: Lamberts Regis R. , Caldenhoven Eric , Lansink Mirian , Witte Gerrit , Vaessen Rob J. , St Cyr John A. , Stienen Ger J. M.
Published: June 2007

During ischemic heart diseases and when heart failure progresses depletion of myocardial energy stores occurs. d-Ribose (R) has been shown to improve cardiac function and energy status after ischemia. Folic acid (FA) is an essential cofactor in the formation of adenine nucleotides. Therefore, we assessed whether chronic R-FA administration during the development of hypertrophy resulted in an improved cardiac function and energy status. In Wistar rats ( n = 40) compensatory right ventricular (RV) hypertrophy was induced by monocrotaline (30 mg/kg; MCT), whereas saline served as control. Both groups received a daily oral dose of either 150 mg·kg−1·day−1dextrose (placebo) or R-FA (150 and 40 mg·kg−1·day−1, respectively). In Langendorff-perfused hearts, RV and left ventricular (LV) pressure development and collagen content as well as total RV adenine nucleotides (TAN), creatine content, and RV and LV collagen content were determined. In the control group R-FA had no effect. In the MCT-placebo group, TAN and creatine content were reduced, RV and LV diastolic pressure-volume relations were steeper, RV systolic pressures were elevated, RV and LV collagen content was increased, and RV-LV diastolic interaction was altered compared with controls. In the MCT-R-FA group, TAN, RV and LV diastolic stiffness, RV and LV collagen content, and RV-LV diastolic interaction were normalized to the values in the control group while creatine content remained depressed and RV systolic function remained elevated. In conclusion, the depression of energy status in compensated hypertrophic myocardium observed was partly prevented by chronic R-FA administration and accompanied by a preservation of diastolic function and collagen deposition.
Viscoelasticity reduces the dynamic stresses and strains in the vessel wall: implications for vessel fatigue
Authors: Zhang Wei , Liu Yi , Kassab Ghassan S.
Published: June 2007

The mechanical behavior of blood vessels is known to be viscoelastic rather than elastic. The functional role of viscoelasticity, however, has remained largely unclear. The hypothesis of this study is that viscoelasticity reduces the stresses and strains in the vessel wall, which may have a significant impact on the fatigue life of the blood vessel wall. To verify the hypothesis, the pulsatile stress in rabbit thoracic artery at physiological loading condition was investigated with a quasi-linear viscoelastic model, where the normalized stress relaxation function is assumed to be isotropic, while the stress-strain relationship is anisotropic and nonlinear. The artery was subjected to the same boundary condition, and the mechanical equilibrium equation was solved for both the viscoelastic and an elastic (which has a constant relaxation function) model. Numerical results show that, compared with purely elastic response, the viscoelastic property of arteries reduces the magnitudes and temporal variations of circumferential stress and strain. The radial wall movement is also reduced due to viscoelasticity. These findings imply that viscoelasticity may be beneficial for the fatigue life of blood vessels, which undergo millions of cyclic mechanical loadings each year of life.
Determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse
Authors: Banerjee Indroneal , Fuseler John W. , Price Robert L. , Borg Thomas K. , Baudino Troy A.
Published: June 2007

Cardiac fibroblasts, myocytes, endothelial cells, and vascular smooth muscle cells are the major cellular constituents of the heart. The aim of this study was to observe alterations in myocardial cell populations during early neonatal development in the adult animal and to observe any variations of the cardiac cell populations in different species, specifically, the rat and mouse. Whole hearts were isolated from either mice or rats during the neonatal and adult stages of development, and single cell suspensions were prepared via sequential collagenase digestion. Heterogeneous cell populations were immunolabeled for specific cell types and analyzed using fluorescence-activated cell sorting (FACS). In addition, the left ventricle, right ventricle, and septa were isolated, fixed, and sectioned for morphometric analyses. These same cardiac regions were also analyzed using FACS. We observed that the adult murine myocardium is composed of ∼56% myocytes, 27% fibroblasts, 7% endothelial cells, and 10% vascular smooth muscle cells. Moreover, our morphometric and FACS data demonstrated similar percentages in the three regions examined. During murine neonatal cardiac development, we observed a marked increase in numbers of cardiac fibroblasts and a resultant decrease in percentages of myocytes in late neonatal development ( day 15). Finally, FACS analyses of the rat heart during development displayed similar results in relation to increases in cardiac fibroblasts during development; however, cell populations in the rat differed markedly from those observed in the mouse. Taken together, these data enabled us to establish a homeostatic model for the myocardium that can be compared with genetic and cardiac disease models.
High tidal volume mechanical ventilation with hyperoxia alters alveolar type II cell adhesion
Authors: Desai Leena P. , Sinclair Scott E. , Chapman Kenneth E. , Hassid Aviv , Waters Christopher M.
Published: June 2007

Patients with acute respiratory distress syndrome undergoing mechanical ventilation may be exposed to both high levels of stretch and high levels of oxygen. We hypothesized that the combination of high stretch and hyperoxia promotes loss of epithelial adhesion and impairs epithelial repair mechanisms necessary for restoration of barrier function. We utilized a model of high tidal volume mechanical ventilation (25 ml/kg) with hyperoxia (50% O2) in rats to investigate alveolar type II (AT2) cell adhesion and focal adhesion signaling. AT2 cells isolated from rats exposed to hyperoxia and high tidal volume mechanical ventilation (MVHO) exhibited significantly decreased cell adhesion and reduction in phosphotyrosyl levels of focal adhesion kinase (FAK) and paxillin compared with control rats, rats exposed to hyperoxia without ventilation (HO), or rats ventilated with normoxia (MV). MV alone increased phosphorylation of p130Cas. RhoA activation was increased by MV, HO, and the combination of MV and HO. Treatment of MVHO cells with keratinocyte growth factor (KGF) for 1 h upon isolation reduced RhoA activity and restored attachment to control levels. Attachment and migration of control AT2 cells was significantly decreased by constitutively active RhoA or a kinase inactive form of FAK (FRNK), whereas expression of dominant negative RhoA in cells from MVHO-treated rats restored cell adhesion. Mechanical ventilation with hyperoxia promotes changes in focal adhesion proteins and RhoA in AT2 cells that may be deleterious for cell adhesion and migration.
Genetic removal of the A2Aadenosine receptor enhances pulmonary inflammation, mucin production, and angiogenesis in adenosine deaminase-deficient mice
Authors: Mohsenin Amir , Mi Tiejuan , Xia Yang , Kellems Rodney E. , Chen Jiang-Fan , Blackburn Michael R.
Published: June 2007

Adenosine is generated at sites of tissue injury where it serves to regulate inflammation and damage. Adenosine signaling has been implicated in the regulation of pulmonary inflammation and damage in diseases such as asthma and chronic obstructive pulmonary disease; however, the contribution of specific adenosine receptors to key immunoregulatory processes in these diseases is still unclear. Mice deficient in the purine catabolic enzyme adenosine deaminase (ADA) develop pulmonary inflammation and mucous metaplasia in association with adenosine elevations making them a useful model for assessing the contribution of specific adenosine receptors to adenosine-mediated pulmonary disease. Studies suggest that the A2Aadenosine receptor (A2AR) functions to limit inflammation and promote tissue protection; however, the contribution of A2AR signaling has not been examined in the ADA-deficient model of adenosine-mediated lung inflammation. The purpose of the current study was to examine the contribution of A2AR signaling to the pulmonary phenotype seen in ADA-deficient mice. This was accomplished by generating ADA/A2AR double knockout mice. Genetic removal of the A2AR from ADA-deficient mice resulted in enhanced inflammation comprised largely of macrophages and neutrophils, mucin production in the bronchial airways, and angiogenesis, relative to that seen in the lungs of ADA-deficient mice with the A2AR. In addition, levels of the chemokines monocyte chemoattractant protein-1 and CXCL1 were elevated, whereas levels of cytokines such as TNF-α and IL-6 were not. There were no compensatory changes in the other adenosine receptors in the lungs of ADA/A2AR double knockout mice. These findings suggest that the A2AR plays a protective role in the ADA-deficient model of pulmonary inflammation.
Interrelations/cross talk between transcellular transport function and paracellular tight junctional properties in lung epithelial and endothelial barriers
Authors: Van Driessche Willy , Kreindler James L. , Malik Asrar B. , Margulies Susan , Lewis Simon A. , Kim Kwang-Jin
Published: June 2007

In this synopsis of a symposium at EB2007, we start with an overview of noise and impedance analyses that have been applied to various epithelial barriers. Noise analysis yields specific information about ion channels and their regulation in epithelial and endothelial barriers. Impedance analysis can yield information about apical and basolateral membrane conductances and paracellular conductance of both epithelial and endothelial barriers. Using a morphologically based model, impedance analysis has been used to assess changes in apical and basolateral membrane surface areas and dimensions of the lateral intercellular space. Impedance analysis of an in vitro airway epithelial barrier under normal, nucleotide-stimulated, and cigarette smoke-exposed conditions yielded information on how activation and inhibition of secretion occur in airway epithelial cells. Similarly, impedance analysis of primary rat alveolar epithelial cell monolayer model under control and EGTA exposure conditions indicate that EGTA causes decreases in resistances of tight junctional routes as well as apical and basolateral cell membranes without causing much change in cell capacitances. In a stretch-caused injury model of alveolar epithelium, transcellular ion transport function and paracellular permeability of solute transport appear to be differentially regulated. Finally, inhibition of caveolae-mediated transcytosis in lung endothelium led to disruption of paracellular routes, increasing the physical dimension and permeability of tight junctional region. These data together demonstrate the cross talk between transcellular and paracellular transport (function and routes) of lung epithelial and endothelial barriers. Mechanistic (e.g., signaling cascades) information on such cross talk remain to be determined.
Role of mitochondrial electron transport complex I in coenzyme Q1reduction by intact pulmonary arterial endothelial cells and the effect of hyperoxia
Authors: Merker Marilyn P. , Audi Said H. , Lindemer Brian J. , Krenz Gary S. , Bongard Robert D.
Published: June 2007

The objective was to determine the impact of intact normoxic and hyperoxia-exposed (95% O2for 48 h) bovine pulmonary arterial endothelial cells in culture on the redox status of the coenzyme Q10homolog coenzyme Q1(CoQ1). When CoQ1(50 μM) was incubated with the cells for 30 min, its concentration in the medium decreased over time, reaching a lower level for normoxic than hyperoxia-exposed cells. The decreases in CoQ1concentration were associated with generation of CoQ1hydroquinone (CoQ1H2), wherein 3.4 times more CoQ1H2was produced in the normoxic than hyperoxia-exposed cell medium (8.2 ± 0.3 and 2.4 ± 0.4 μM, means ± SE, respectively) after 30 min. The maximum CoQ1reduction rate for the hyperoxia-exposed cells, measured using the cell membrane-impermeant redox indicator potassium ferricyanide, was about one-half that of normoxic cells (11.4 and 24.1 nmol·min−1·mg−1cell protein, respectively). The mitochondrial electron transport complex I inhibitor rotenone decreased the CoQ1reduction rate by 85% in the normoxic cells and 44% in the hyperoxia-exposed cells. There was little or no inhibitory effect of NAD(P)H:quinone oxidoreductase 1 (NQO1) inhibitors on CoQ1reduction. Intact cell oxygen consumption rates and complex I activities in mitochondria-enriched fractions were also lower for hyperoxia-exposed than normoxic cells. The implication is that intact pulmonary endothelial cells influence the redox status of CoQ1via complex I-mediated reduction to CoQ1H2, which appears in the extracellular medium, and that the hyperoxic exposure decreases the overall CoQ1reduction capacity via a depression in complex I activity.