bluuebunny/crossref_metadata_2025 huggingface dataset:
Ammonia-Induced Formation of an AmtB-GlnK Complex Is Not Sufficient for Nitrogenase Regulation in the Photosynthetic BacteriumRhodobacter capsulatus
Authors: Tremblay Pier-Luc , Hallenbeck Patrick C.
Published: December 2007

A series ofRhodobacter capsulatusAmtB variants were created and assessed for effects on ammonia transport, formation of AmtB-GlnK complexes, and regulation of nitrogenase activity and NifH ADP-ribosylation. Confirming previous reports, H193 and H342 were essential for ammonia transport and the replacement of aspartate 185 with glutamate reduced ammonia transport. Several amino acid residues, F131, D334, and D335, predicted to be critical for AmtB activity, are shown here for the first time by mutational analysis to be essential for transport. Alterations of the C-terminal tail reduced methylamine transport, prevented AmtB-GlnK complex formation, and abolished nitrogenase switch-off and NifH ADP-ribosylation. On the other hand, D185E, with a reduced level of transport, was capable of forming an ammonium-induced complex with GlnK and regulating nitrogenase. This reinforces the notions that ammonia transport is not sufficient for nitrogenase regulation and that formation of an AmtB-GlnK complex is necessary for these processes. However, some transport-incompetent AmtB variants, i.e., F131A, H193A, and H342A, form ammonium-induced complexes with GlnK but fail to properly regulate nitrogenase. These results show that formation of an AmtB-GlnK complex is insufficient in itself for nitrogenase regulation and suggest that partial ammonia transport or occupation of the pore by ammonia is essential for this function.
The LysR-Type Transcriptional Regulator LeuO Controls Expression of Several Genes in
Salmonella enterica
Serovar Typhi
Authors: Hernández-Lucas I. , Gallego-Hernández A. L. , Encarnación S. , Fernández-Mora M. , Martínez-Batallar A. G. , Salgado H. , Oropeza R. , Calva E.
Published: December 2007

LeuO is a LysR-type transcriptional regulator that has been implicated in the bacterial stringent response and in the virulence of
Salmonella
. A genomic analysis with
Salmonella enterica
serovar Typhi revealed that LeuO is a positive regulator of OmpS1, OmpS2, AssT, and STY3070. In contrast, LeuO down-regulated the expression of OmpX, Tpx, and STY1978. Transcriptional fusions supported the positive and negative LeuO regulation. Expression of
ompS1
,
assT
, and STY3070 was induced in an
hns
mutant, consistent with the notion that H-NS represses these genes; transcriptional activity was lower for
tpx
and STY1978 in an
hns
background, suggesting that this global regulatory protein has a positive effect. In contrast,
ompS2
and
ompX
expression appeared to be H-NS independent. LeuO specifically bound to the 5′ intergenic regions of
ompS2
,
assT
, STY3070,
ompX
, and
tpx
, while it was not observed to bind to the promoter region of STY1978, suggesting that LeuO regulates in direct and indirect ways. In this work, a novel set of genes belonging to the LeuO regulon are described; interestingly, these genes are involved in a variety of biological processes, suggesting that LeuO is a global regulator in
Salmonella
.
LuxG Is a Functioning Flavin Reductase for Bacterial Luminescence
Authors: Nijvipakul Sarayut , Wongratana Janewit , Suadee Chutintorn , Entsch Barrie , Ballou David P. , Chaiyen Pimchai
Published: December 2007

The
luxG
gene is part of the
lux
operon of marine luminous bacteria.
luxG
has been proposed to be a flavin reductase that supplies reduced flavin mononucleotide (FMN) for bacterial luminescence. However, this role has never been established because the gene product has not been successfully expressed and characterized. In this study,
luxG
from
Photobacterium leiognathi
TH1 was cloned and expressed in
Escherichia coli
in both native and C-terminal His
6
-tagged forms. Sequence analysis indicates that the protein consists of 237 amino acids, corresponding to a subunit molecular mass of 26.3 kDa. Both expressed forms of LuxG were purified to homogeneity, and their biochemical properties were characterized. Purified LuxG is homodimeric and has no bound prosthetic group. The enzyme can catalyze oxidation of NADH in the presence of free flavin, indicating that it can function as a flavin reductase in luminous bacteria. NADPH can also be used as a reducing substrate for the LuxG reaction, but with much less efficiency than NADH. With NADH and FMN as substrates, a Lineweaver-Burk plot revealed a series of convergent lines characteristic of a ternary-complex kinetic model. From steady-state kinetics data at 4°C pH 8.0,

K
m

for NADH,

K
m

for FMN, and
k
cat
were calculated to be 15.1 μM, 2.7 μM, and 1.7 s
−1
, respectively. Coupled assays between LuxG and luciferases from
P. leiognathi
TH1 and
Vibrio campbellii
also showed that LuxG could supply FMNH
−
for light emission in vitro. A
luxG
gene knockout mutant of
P. leiognathi
TH1 exhibited a much dimmer luminescent phenotype compared to the native
P. leiognathi
TH1, implying that LuxG is the most significant source of FMNH
−
for the luminescence reaction in vivo.
Genetic Analysis of Lipooligosaccharide Core Biosynthesis in
Campylobacter jejuni
81-176
Authors: Kanipes Margaret I. , Tan Xuan , Akelaitis Aurel , Li Jiajun , Rockabrand David , Guerry Patricia , Monteiro Mario A.
Published: December 2007

We report isolation and characterization of
Campylobacter jejuni
81-176
lgtF
and
galT
lipooligosaccharide (LOS) core mutants. It has been suggested that the
lgtF
gene of
C. jejuni
encodes a two-domain glucosyltransferase that is responsible for the transfer of a β-1,4-glucose residue on heptosyltransferase I (Hep I) and for the transfer of a β-1,2-glucose residue on Hep II. A site-specific mutation in the
lgtF
gene of
C. jejuni
81-176 resulted in expression of a truncated LOS, and complementation of the mutant in
trans
restored the core mobility to that of the wild type. Mass spectrometry and nuclear magnetic resonance of the truncated LOS confirmed the loss of two glucose residues, a β-1,4-glucose on Hep I and a β-1,2-glucose on Hep II. Mutation of another gene,
galT
, encoding a glycosyltransferase, which maps outside the region defined as the LOS biosynthetic locus in
C. jejuni
81-176, resulted in loss of the β-(1,4)-galactose residue and all distal residues in the core. Both mutants invaded intestinal epithelial cells in vitro at levels comparable to the wild-type levels, in marked contrast to a deeper inner core
waaC
mutant. These studies have important implications for the role of LOS in the pathogenesis of
Campylobacter
-mediated infection.
lfnA
from
Pseudomonas aeruginosa
O12 and
wbuX
from
Escherichia coli
O145 Encode Membrane-Associated Proteins and Are Required for Expression of 2,6-Dideoxy-2-Acetamidino-
l
-Galactose in Lipopolysaccharide O Antigen
Authors: King Jerry D. , Mulrooney Erin F. , Vinogradov Evgeny , Kneidinger Bernd , Mead Kristen , Lam Joseph S.
Published: December 2007

The rare sugar 2,6-dideoxy-2-acetamidino-
l
-galactose (
l
-FucNAm) is found only in bacteria and is a component of cell surface glycans in a number of pathogenic species, including the O antigens of
Pseudomonas aeruginosa
serotype O12 and
Escherichia coli
O145.
P
.
aeruginosa
is an important opportunistic pathogen, and the O12 serotype is associated with multidrug-resistant epidemic outbreaks. O145 is one of the classic non-O157 serotypes associated with Shiga toxin-producing, enterohemorrhagic
E
.
coli
. The acetamidino (NAm) moiety of
l
-FucNAm is of interest, because at neutral pH it contributes a positive charge to the cell surface, and we aimed to characterize the biosynthesis of this functional group. The pathway is not known, but expression of NAm-modified sugars coincides with the presence of a
pseA
homologue in the relevant biosynthetic locus. PseA is a putative amidotransferase required for synthesis of a NAm-modified sugar in
Campylobacter jejuni
. In
P
.
aeruginosa
O12 and
E
.
coli
O145, the
pseA
homologues are
lfnA
and
wbuX
, respectively, and we hypothesized that these genes function in
l
-FucNAm biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and nuclear magnetic resonance analysis of the
lfnA
mutant O-antigen structure indicated that the mutant expresses 2,6-dideoxy-2-acetamido-
l
-galactose (
l
-FucNAc) in place of
l
-FucNAm. The mutation could be complemented by expression of either His
6
-tagged
lfnA
or
wbuX
in
trans
, confirming that these genes are functional homologues and that they are required for NAm moiety synthesis. Both proteins retained their activity when fused to a His
6
tag and localized to the membrane fraction. These data will assist future biochemical investigation of this pathway.
Altered Utilization ofN-Acetyl-d-Galactosamine byEscherichia coliO157:H7 from the 2006 Spinach Outbreak
Authors: Mukherjee Amit , Mammel Mark K. , LeClerc J. Eugene , Cebula Thomas A.
Published: December 2007

In silico analyses of previously sequenced strains ofEscherichia coliO157:H7, EDL933 and Sakai, localized the gene cluster for the utilization ofN-acetyl-d-galactosamine (Aga) andd-galactosamine (Gam). This gene cluster encodes the Aga phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) and other catabolic enzymes responsible for transport and catabolism of Aga. As the complete coding sequences for enzyme IIA (EIIA)Aga/Gam, EIIBAga, EIICAga, and EIIDAgaof the Aga PTS are present,E. coliO157:H7 strains normally are able to utilize Aga as a sole carbon source. The Gam PTS complex, in contrast, lacks EIICGam, and consequently,E. coliO157:H7 strains cannot utilize Gam. Phenotypic analyses of 120 independent isolates ofE. coliO157:H7 from our culture collection revealed that the overwhelming majority (118/120) displayed the expected Aga+Gam−phenotype. Yet, when 194 individual isolates, derived from a 2006 spinach-associatedE. coliO157:H7 outbreak, were analyzed, all (194/194) displayed an Aga−Gam−phenotype. Comparison ofaga/gamsequences from two spinach isolates with those of EDL933 and Sakai revealed a single nucleotide change (G:C→A:T) in theagaFgene in the spinach-associated isolates. The base substitution inagaF, which encodes EIIAAga/Gamof the PTS, changes a conserved glycine residue to serine (Gly91Ser). Pyrosequencing of this region showed that all spinach-associatedE. coliO157:H7 isolates harbored this same G:C→A:T substitution. Notably, whenagaF+was cloned into an expression vector and transformed into six spinach isolates, all (6/6) were able to grow on Aga, thus demonstrating that the Gly91Ser substitution underlies the Aga−phenotype in these isolates.
Diagnostic and prognostic significance of cyclin A expression in low-grade astrocytomas: comparison with astrogliosis and high-grade tumours
Authors: Hara A , Saegusa M , Ichinoe M , Okayasu I
Published: December 2007

Aim:

Definitive distinction between low-grade astrocytoma and astrogliosis is a long-standing difficulty due to their similar histopathological characteristics. To clarify differences in biological significance, this study focused on various components of the cell cycle machinery and proliferation as key parameters, comparing expression in astrogliosis, as well as low- and high-grade astrocytomas.

Methods:

The expression of p16, p21 and p27, and cyclin A, cyclin D1, cyclin E, Rb and Ki-67 was immunohistochemically examined in 40 cases of astrogliosis and 48 cases of low-grade astrocytomas (grade II), as well as 50 high-grade tumours (grades III and IV). The results were also compared with survival data for the astrocytomas.

Results:

Cell proliferation determined by Ki-67 immunoreactivity did not differ between astrogliosis and low-grade tumours. Average labelling indices (LIs) for p16, p21, Rb, cyclin A and cyclin E showed a stepwise increase from astrogliosis, through low- to high-grade astrocytomas, indicating the possibility that over 9%, 6% and 4% of LIs for p16, p21 and cyclin A, respectively, may be useful predictors in the case of the latter, in contrast to significant decrease in p27 LIs. Significantly higher mean LI values for cyclin D1 were also evident in astrogliosis (12.42) as compared with astrocytomas (low grade, 2.26; high grade, 4.60). Positive correlations between LIs for Rb and Ki-67 were observed with astrogliosis and low- but not high-grade tumours. In addition, high cyclin A LI values were independently associated with poor outcome in low-grade tumours.

Conclusion:

These findings provide evidence that expression of cell-cycle-related molecules may be a reliable parameter for differential diagnosis of low-grade astrocytomas and astrogliosis. Moreover, detection of cyclin A appears to be useful for predicting behaviour of low-grade astrocytomas.
Reduction of E-cadherin expression is associated with non-lobular breast carcinomas of basal-like and triple negative phenotype
Authors: Mahler-Araujo B , Savage K , Parry S , Reis-Filho J S
Published: December 2007

Aim:

E-cadherin inactivation in breast cancer has been shown to be strongly associated with lobular breast cancer. However, little is known about the levels of E-cadherin expression according to the breast cancer “molecular” subtypes. The aim of this study was to address the distribution of E-cadherin expression according to the different molecular subtypes of breast cancer.

Methods:

E-cadherin expression was immunohistochemically analysed in a tissue microarray containing duplicate cores of 245 invasive breast carcinomas, of which 182 cases were of non-lobular histology, using a semi-quantitative scoring system based on the percentage of cells showing membrane immunopositivity.

Results:

In non-lobular breast carcinomas, reduced and/or negative E-cadherin expression was significantly associated with lack of oestrogen receptor expression, low levels ofCCND1expression, positivity for cytokeratins 5/6 and 17, epidermal growth factor receptor and caveolins 1 and 2, p53 expression, high MIB-1 proliferation indices, basal-like phenotype and triple negative phenotype.

Conclusion:

This study demonstrates that in the group of non-lobular breast cancers, reduction/lack of E-cadherin expression is preferentially found in basal-like breast carcinomas.
Determinants of coronary microvascular dysfunction in symptomatic hypertrophic cardiomyopathy
Authors: Knaapen Paul , Germans Tjeerd , Camici Paolo G. , Rimoldi Ornella E. , ten Cate Folkert J. , ten Berg Jurrien M. , Dijkmans Pieter A. , Boellaard Ronald , van Dockum Willem G. , Götte Marco J. W. , Twisk Jos W. R. , van Rossum Albert C. , Lammertsma Adriaan A. , Visser Frans C.
Published: December 2007

Impaired hyperemic myocardial blood flow (MBF) in hypertrophic cardiomyopathy (HCM), despite normal epicardial coronary arteries, results in microvascular dysfunction. The aim of the present study was to determine the relative contribution of extravascular compressive forces to microvascular dysfunction in HCM. Eighteen patients with symptomatic HCM and normal coronary arteries and 10 age-matched healthy volunteers were studied with PET to quantify resting and hyperemic MBF at a subendocardial and subepicardial level. In HCM patients, MRI was performed to determine left ventricular (LV) mass index (LVMI) and volumes, echocardiography to assess diastolic perfusion time, heart catheterization to measure LV outflow tract gradient (LVOTG) and LV pressures, and serum NH2-terminal pro-brain natriuretic peptide (NT-proBNP) as a biochemical marker of LV wall stress. Hyperemic MBF was blunted in HCM vs. controls (2.26 ± 0.97 vs. 2.93 ± 0.64 ml·min−1·g−1, P < 0.05). In contrast to controls (1.38 ± 0.15 to 1.25 ± 0.19, P = not significant), the endocardial-to-epicardial MBF ratio decreased significantly in HCM during hyperemia (1.20 ± 0.11 to 0.88 ± 0.18, P < 0.01). This pattern was similar for hypertrophied septum and lateral wall. Hyperemic MBF was inversely correlated with LVOTG, NT-proBNP, left atrial volume index, and LVMI (all P < 0.01). Multivariate regression analysis, however, revealed that only LVMI and NT-proBNP were independently related to hyperemic MBF, with greater impact at the subendocardial myocardial layer. Hyperemic MBF is more severely impaired at the subendocardial level in HCM patients. The level of impairment is related to markers of increased hemodynamic LV loading conditions and LV mass. These observations suggest that, in addition to reduced capillary density caused by hypertrophy, extravascular compressive forces contribute to microvascular dysfunction in HCM patients.
Go controls the hyperpolarization-activated current in embryonic stem cell-derived cardiocytes
Authors: Ye Chian P. , Duan Sheng Zhong , Milstone David S. , Mortensen Richard M.
Published: December 2007

Hyperpolarization current ( If) is an important player in controlling heart rate and is stimulated by cAMP and inhibited by members of the pertussis toxin-sensitive G-protein Gi/Go family. We have successfully derived cardiocytes from embryonic stem cells lacking Go or Gi2 and Gi3. We have established that both basal and isoproterenol-stimulated activities of If in these cardiocytes have typical nodal-atrial characteristics and are unaffected by targeted gene inactivation of the G proteins Go or Gi2 and Gi3. Under basal conditions, both Go and Gi are required for muscarinic inhibition of If activity via a mechanism that involves the generation of nitric oxide, whereas, with prior stimulation by β-agonists, only Go is required and Gi and nitric oxide production are not. Our findings establish an essential role for Go in the antiadrenergic effect of muscarinic agent on If.
Microtopography and flow modulate the direction of endothelial cell migration
Authors: Uttayarat P. , Chen M. , Li M. , Allen F. D. , Composto R. J. , Lelkes P. I.
Published: December 2007

The migration of vascular endothelial cells under flow can be modulated by the addition of chemical or mechanical stimuli. The aim of this study was to investigate how topographic cues derived from a substrate containing three-dimensional microtopography interact with fluid shear stress in directing endothelial cell migration. Subconfluent bovine aortic endothelial cells were seeded on fibronectin-coated poly(dimethylsiloxane) substrates patterned with a combinatorial array of parallel and orthogonal microgrooves ranging from 2 to 5 μm in width at a constant depth of 1 μm. During a 4-h time-lapse observation in the absence of flow, the majority of the prealigned cells migrated parallel to the grooves with the distribution of their focal adhesions (FAs) depending on the groove width. No change in this migratory pattern was observed after the cells were exposed to moderate shear stress (13.5 dyn/cm2), irrespective of groove direction with respect to flow. After 4-h exposure to high shear stress (58 dyn/cm2) parallel to the grooves, the cells continued to migrate in the direction of both grooves and flow. By contrast, when microgrooves were oriented perpendicular to flow, most cells migrated orthogonal to the grooves and downstream with flow. Despite the change in the migration direction of the cells under high shear stress, most FAs and actin microfilaments maintained their original alignment parallel to the grooves, suggesting that topographic cues were more effective than those derived from shear stress in guiding the orientation of cytoskeletal and adhesion proteins during the initial exposure to flow.
Hypoxic preconditioning increases survival and angiogenic potency of peripheral blood mononuclear cells via oxidative stress resistance
Authors: Kubo Masayuki , Li Tao-Sheng , Suzuki Ryo , Shirasawa Bungo , Morikage Noriyasu , Ohshima Mako , Qin Shu-Lan , Hamano Kimikazu
Published: December 2007

Cell-based angiogenesis is a promising treatment for ischemic diseases; however, the survival of implanted cells is impaired by oxidative stress in the ischemic microenvironment. We tested the hypothesis that hypoxic preconditioning of implanted cells enhances their resistance against oxidative stress, increasing cell survival and angiogenic potency after implantation into ischemic tissue. Mouse peripheral blood mononuclear cells (PBMNCs) were collected and subjected to hypoxic preconditioning by culture for 24 h in 2% O2 at 33°C. Hypoxic preconditioning of PBMNCs increased the expression of various genes related to antioxidant and survival signals remarkably. Compared with cells cultured under normoxia, the hypoxia-preconditioned PBMNCs showed significantly lower reactive oxygen species (ROS) accumulation and higher cell survival under oxidative stress induced by LY-83583 (a superoxide generator). Three days after intramuscular implantation into the ischemic hindlimbs of mice, survival of the hypoxia-preconditioned PBMNCs was high, whereas that of the normoxia-cultured PBMNCs was relatively low. Furthermore, 28 days after treatment microvessel density and blood flow in the ischemic hindlimbs were significantly better in the mice implanted with hypoxia-preconditioned PBMNCs than in those implanted with normoxia-cultured PBMNCs. Hypoxic preconditioning increased the survival and angiogenic potency of PBMNCs, through oxidative stress resistance mechanisms.
Genes overexpressed in cerebral arteries following salt-induced hypertensive disease are regulated by angiotensin II, JunB, and CREB
Authors: Rose Patricia , Bond Jeffrey , Tighe Scott , Toth Michael J. , Wellman Theresa L. , de Montiano Eva Maria Briso , Lewinter Martin M. , Lounsbury Karen M.
Published: December 2007

Although changes in gene expression are necessary for arterial remodeling during hypertension, the genes altered and their mechanisms of regulation remain uncertain. The goal of this study was to identify cerebral artery genes altered by hypertension and define signaling pathways important in their regulation. Intact cerebral arteries from Dahl salt-sensitive normotensive and hypertensive high-salt (HS) rats were examined by immunostaining, revealing an increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and expression of the proliferative marker Ki-67 in arteries from hypertensive animals. Arterial RNA analyzed by microarray and validated with RT-quantitative PCR revealed that jun family member junB and matricellular genes plasminogen activator inhibitor-1 (PAI-1) and osteopontin (OPN) were significantly overexpressed in HS arteries. Fisher exact test and annotation-based gene subsets showed that genes upregulated by Jun and Ca2+/cAMP-response element-binding protein (CREB) were overrepresented. A model of cultured rat cerebrovascular smooth muscle cells was used to test the hypothesis that angiotensin II (ANG II), JunB, and CREB are important in the regulation of genes identified in the rat hypertension model. ANG II induced a transient induction of junB and a delayed induction of PAI-1 and OPN mRNA levels, which were reduced by ERK inhibition with U-0126. Silencing junB using small-interfering RNA reduced mRNA levels of OPN but not PAI-1. The silencing of CREB reduced PAI-1 induction by ANG II but enhanced the transcription of OPN. Together, these results suggest that salt-induced hypertensive disease promotes changes in matricellular genes that are stimulated by ANG II, regulated by ERK, and selectively regulated by JunB and CREB.
Heterogeneous transmural proteoglycan distribution provides a mechanism for regulating residual stresses in the aorta
Authors: Azeloglu Evren U. , Albro Michael B. , Thimmappa Vikrum A. , Ateshian Gerard A. , Costa Kevin D.
Published: December 2007

The arterial wall contains a significant amount of charged proteoglycans, which are inhomogeneously distributed, with the greatest concentrations in the intimal and medial layers. The hypothesis of this study is that the transmural distribution of proteoglycans plays a significant role in regulating residual stresses in the arterial wall. This hypothesis was first tested theoretically, using the framework of mixture theory for charged hydrated tissues, and then verified experimentally by measuring the opening angle of rat aorta in NaCl solutions of various ionic strengths. A three-dimensional finite element model of aortic ring, using realistic values of the solid matrix shear modulus and proteoglycan fixed-charge density, yielded opening angles and changes with osmolarity comparable to values reported in the literature. Experimentally, the mean opening angle in isotonic saline (300 mosM) was 15 ± 17° and changed to 4 ± 19° and 73 ± 18° under hypertonic (2,000 mosM) and hypotonic (0 mosM) conditions, respectively ( n = 16). In addition, the opening angle in isotonic (300 mosM) sucrose, an uncharged molecule, was 60 ± 16° ( n = 11), suggesting that the charge effect, not cellular swelling, was the major underlying mechanism for these observations. The extent of changes in opening angle under osmotic challenges suggests that transmural heterogeneity of fixed-charge density plays a crucial role in governing the zero-stress configuration of the aorta. A significant implication of this finding is that arterial wall remodeling in response to altered wall stresses may occur via altered deposition of proteoglycans across the wall thickness, providing a novel mechanism for regulating mechanical homeostasis in vascular tissue.
A profound decrease in maternal arginine uptake provokes endothelial nitration in the pregnant rat
Authors: Reshef Ran , Schwartz Doron , Ingbir Merav , Shtabsky Alexander , Chernichovski Tamara , Isserlin Benjamin A. , Chernin Gil , Levo Yoram , Schwartz Idit F.
Published: December 2007

While a specific role for nitric oxide (NO) in inducing the hemodynamic alterations of pregnancy is somewhat controversial, it is widely accepted that excess NO is generated during pregnancy. l-Arginine is the sole precursor for NO biosynthesis. Among several transporters that mediate l-arginine uptake, cationic amino acid transporter-1 (CAT-1) acts as the specific arginine transporter for endothelial NO synthase. The present study was designed to test the hypothesis that, during pregnancy, when arginine consumption by the fetus is significantly increased, compensatory changes in maternal arginine uptake affect the endothelium. Uptake of radiolabeled arginine (l-[3H]arginine) by freshly harvested maternal aortic rings from pregnant rats decreased by 65 and 30% in mid- and late pregnancy, respectively, compared with those obtained from virgin animals. This decrease was associated with a significant increase in endothelial protein nitration (the footprint of peroxynitrite generation), as shown by both Western blotting and immunohistochemistry utilizing anti-nitrotyrosine antibodies, reflecting endothelial damage. Northern blot analysis revealed that steady-state aortic CAT-1 mRNA levels did not change throughout pregnancy, whereas CAT-1 protein abundance was significantly increased, peaking at mid-pregnancy. Protein content of protein kinase C (PKC)-α, which was previously shown to decrease CAT-1 activity, increased significantly in the pregnant animals and was associated with a significant increase in CAT-1 phosphorylation. Intraperitoneal injection of α-tocopherol, a PKC-α inhibitor, prevented the decrease in arginine transport and attenuated protein nitration. In conclusion, aortic arginine uptake is reduced during pregnancy, through posttranslational modulation of CAT-1 protein, presumably via upregulation of PKC-α. The aforementioned findings are associated with an increase in protein nitration and, therefore, in selected individuals, may lead to the development of certain forms of endothelial dysfunction, like preeclampsia.
Role of asymmetric dimethylarginine for angiotensin II-induced target organ damage in mice
Authors: Jacobi Johannes , Maas Renke , Cordasic Nada , Koch Kilian , Schmieder Roland E. , Böger Rainer H. , Hilgers Karl F.
Published: December 2007

The aim of the present study was to investigate the role of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) and its degrading enzyme dimethylarginine dimethylaminohydrolase (DDAH) in angiotensin II (ANG II)-induced hypertension and target organ damage in mice. Mice transgenic for the human DDAH1 gene (TG) and wild-type (WT) mice (each, n = 28) were treated with 1.0 μg·kg−1·min−1 ANG II, 3.0 μg·kg−1·min−1 ANG II, or phosphate-buffered saline over 4 wk via osmotic minipumps. Blood pressure, as measured by tail cuff, was elevated to the same degree in TG and WT mice. Plasma levels of ADMA were lower in TG than WT mice and were not affected after 4 wk by either dose of ANG II in both TG and WT animals. Oxidative stress within the wall of the aorta, measured by fluorescence microscopy using the dye dihydroethidium, was significantly reduced in TG mice. ANG II-induced glomerulosclerosis was similar between WT and TG mice, whereas renal interstitial fibrosis was significantly reduced in TG compared with WT animals. Renal mRNA expression of protein arginine methyltransferase (PRMT)1 and DDAH2 increased during the infusion of ANG II, whereas PRMT3 and endogenous mouse DDAH1 expression remained unaltered. Chronic infusion of ANG II in mice has no effect on the plasma levels of ADMA after 4 wk. However, an overexpression of DDAH1 alleviates ANG II-induced renal interstitial fibrosis and vascular oxidative stress, suggesting a blood pressure-independent effect of ADMA on ANG II-induced target organ damage.
20-Hydroxyeicosatetraenoic acid causes endothelial dysfunction via eNOS uncoupling
Authors: Cheng Jennifer , Ou Jing-Song , Singh Harpreet , Falck John R. , Narsimhaswamy Dubasi , Pritchard Kirkwood A. , Schwartzman Michal Laniado
Published: December 2007

Nitric oxide (NO), generated from l-arginine by endothelial nitric oxide synthase (eNOS), is a key endothelial-derived factor whose bioavailability is essential to the normal function of the endothelium. Endothelium dysfunction is characterized by loss of NO bioavailability because of either reduced formation or accelerated degradation of NO. We have recently reported that overexpression of vascular cytochrome P-450 (CYP) 4A in rats caused hypertension and endothelial dysfunction driven by increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), a major vasoconstrictor eicosanoid in the microcirculation. To further explore cellular mechanisms underlying CYP4A-20-HETE-driven endothelial dysfunction, the interactions between 20-HETE and the eNOS-NO system were examined in vitro. Addition of 20-HETE to endothelial cells at concentrations as low as 1 nM reduced calcium ionophore-stimulated NO release by 50%. This reduction was associated with a significant increase in superoxide production. The increase in superoxide in response to 20-HETE was prevented by NG-nitro-l-arginine methyl ester, suggesting that uncoupled eNOS is a source of this superoxide. The response to 20-HETE was specific in that 19-HETE did not affect NO or superoxide production, and, in fact, the response to 20-HETE could be competitively antagonized by 19(R)-HETE. 20-HETE had no effect on phosphorylation of eNOS protein at serine-1179 or threonine-497 following addition of calcium ionophore; however, 20-HETE inhibited association of eNOS with 90-kDa heat shock protein (HSP90). In vivo, impaired acetylcholine-induced relaxation in arteries overexpressing CYP4A was associated with a marked reduction in the levels of phosphorylated vasodilator-stimulated phosphoprotein, an indicator of bioactive NO, that was reversed by inhibition of 20-HETE synthesis or action. Because association of HSP90 with eNOS is critical for eNOS activation and coupled enzyme activity, inhibition of this association by 20-HETE may underlie the mechanism, at least in part, by which increased CYP4A expression and activity cause endothelial dysfunction.
Neonatal rat cardiac fibroblasts express three types of voltage-gated K+ channels: regulation of a transient outward current by protein kinase C
Authors: Walsh Kenneth B. , Zhang Jining
Published: December 2007

Cardiac fibroblasts regulate myocardial development via mechanical, chemical, and electrical interactions with associated cardiomyocytes. The goal of this study was to identify and characterize voltage-gated K+ (Kv) channels in neonatal rat ventricular fibroblasts. With the use of the whole cell arrangement of the patch-clamp technique, three types of voltage-gated, outward K+ currents were measured in the cultured fibroblasts. The majority of cells expressed a transient outward K+ current ( Ito) that activated at potentials positive to −40 mV and partially inactivated during depolarizing voltage steps. Ito was inhibited by the antiarrhythmic agent flecainide (100 μM) and BaCl2 (1 mM) but was unaffected by 4-aminopyridine (4-AP; 0.5 and 1 mM). A smaller number of cells expressed one of two types of kinetically distinct, delayed-rectifier K+ currents [ IK fast ( IKf) and IK slow ( IKs)] that were strongly blocked by 4-AP. Application of phorbol 12-myristate 13-acetate, to stimulate protein kinase C (PKC), inhibited Ito but had no effect on IKf and IKs. Immunoblot analysis revealed the presence of Kv1.4, Kv1.2, Kv1.5, and Kv2.1 α-subunits but not Kv4.2 or Kv1.6 α-subunits in the fibroblasts. Finally, pretreatment of the cells with 4-AP inhibited angiotensin II-induced intracellular Ca2+ mobilization. Thus neonatal cardiac fibroblasts express at least three different Kv channels that may contribute to electrical/chemical signaling in these cells.
Disruption of the circadian clock within the cardiomyocyte influences myocardial contractile function, metabolism, and gene expression
Authors: Bray Molly S. , Shaw Chad A. , Moore Michael W. S. , Garcia Rodrigo A. P. , Zanquetta Melissa M. , Durgan David J. , Jeong William J. , Tsai Ju-Yun , Bugger Heiko , Zhang Dongfang , Rohrwasser Andreas , Rennison Julie H. , Dyck Jason R. B. , Litwin Sheldon E. , Hardin Paul E. , Chow Chi-Wing , Chandler Margaret P. , Abel E. Dale , Young Martin E.
Published: December 2007

Virtually every mammalian cell, including cardiomyocytes, possesses an intrinsic circadian clock. The role of this transcriptionally based molecular mechanism in cardiovascular biology is poorly understood. We hypothesized that the circadian clock within the cardiomyocyte influences diurnal variations in myocardial biology. We, therefore, generated a cardiomyocyte-specific circadian clock mutant (CCM) mouse to test this hypothesis. At 12 wk of age, CCM mice exhibit normal myocardial contractile function in vivo, as assessed by echocardiography. Radiotelemetry studies reveal attenuation of heart rate diurnal variations and bradycardia in CCM mice (in the absence of conduction system abnormalities). Reduced heart rate persisted in CCM hearts perfused ex vivo in the working mode, highlighting the intrinsic nature of this phenotype. Wild-type, but not CCM, hearts exhibited a marked diurnal variation in responsiveness to an elevation in workload (80 mmHg plus 1 μM epinephrine) ex vivo, with a greater increase in cardiac power and efficiency during the dark (active) phase vs. the light (inactive) phase. Moreover, myocardial oxygen consumption and fatty acid oxidation rates were increased, whereas cardiac efficiency was decreased, in CCM hearts. These observations were associated with no alterations in mitochondrial content or structure and modest mitochondrial dysfunction in CCM hearts. Gene expression microarray analysis identified 548 and 176 genes in atria and ventricles, respectively, whose normal diurnal expression patterns were altered in CCM mice. These studies suggest that the cardiomyocyte circadian clock influences myocardial contractile function, metabolism, and gene expression.
All-transretinoic acid prevents development of cardiac remodeling in aortic banded rats by inhibiting the renin-angiotensin system
Authors: Choudhary Rashmi , Palm-Leis Ants , Scott Robert C. , Guleria Rakeshwar S. , Rachut Eric , Baker Kenneth M. , Pan Jing
Published: December 2007

This study was designed to determine the effect of all- trans retinoic acid (RA) on the development of cardiac remodeling in a pressure overload rat model. Male Sprague-Dawley rats were subjected to sham operation and the aortic constriction procedure. A subgroup of sham control and aortic constricted rats were treated with RA for 5 mo after surgery. Pressure-overloaded rats showed significantly increased interstitial and perivascular fibrosis, heart weight-to-body weight ratio, and gene expression of atrial natriuretic peptide and brain natriuretic peptide. Echocardiographic analysis showed that pressure overload induced systolic and diastolic dysfunction, as evidenced by decreased fractional shortening, ejection fraction, stroke volume, and increased E-to-Earatio and isovolumic relaxation time. RA treatment prevented the above changes in cardiac structure and function and hypertrophic gene expression in pressure-overloaded rats. RA restored the ratio of Bcl-2 to Bax, inhibited cleavage of caspase-3 and -9, and prevented the decreases in the levels of SOD-1 and SOD-2. Pressure overload-induced phosphorylation of ERK1/2, JNK, and p38 was inhibited by RA, via upregulation of mitogen-activated protein kinase phosphatase (MKP)-1 and MKP-2. The pressure overload-induced production of angiotensin II was inhibited by RA via upregulation of expression of angiotensin-converting enzyme (ACE)2 and through inhibition of the expression of cardiac and renal renin, angiotensinogen, ACE, and angiotensin type 1 receptor. Similar results were observed in cultured neonatal cardiomyocytes in response to static stretch. These results demonstrate that RA has a significant inhibitory effect on pressure overload-induced cardiac remodeling, through inhibition of the expression of renin-angiotensin system components.
IL-1β, BK, and TGF-β1attenuate PGI2-mediated cAMP formation in human pulmonary artery smooth muscle cells by multiple mechanisms involving p38 MAP kinase and PKA
Authors: El-Haroun H. , Clarke D. L. , Deacon K. , Bradbury D. , Clayton A. , Sutcliffe A. , Knox Alan J.
Published: December 2007

We have previously shown that interleukin (IL)-1β, transforming growth factor (TGF)-β1, or bradykinin (BK) impair cAMP generation in response to prostacyclin analogs in human pulmonary artery smooth muscle (PASM), suggesting that inflammation can impair the effects of prostacyclin analogs on PASM in pulmonary hypertension. Here we explored the biochemical mechanisms involved. We found that IL-1β, BK, and TGF-β1reduced adenylyl cyclase isoform 1, 2, and 4 mRNA, increased Gαiprotein levels, and reduced prostacyclin receptor (IP receptor) mRNA expression. In contrast, Gαsprotein levels were unchanged. Protein kinase A (PKA) (H-89, KT-2750, PKIm) and p38 mitogen-activated protein (MAP) kinase (SB-202190) inhibitors attenuated these effects, but protein kinase C (bisindolylmaleide) or phosphoinositol 3-kinase (LY-294002) inhibitors did not. Fluorescent kemptide assay and Western blotting confirmed that PKA and p38 MAP kinase were activated by IL-1β, BK, and TGF-β1. These studies suggest that IL-1β, BK, and TGF-β1impair IP receptor-mediated cAMP accumulation by multiple effects on different components of the signaling pathway and that these effects are PKA and p38 MAP kinase dependent.
Identification of Nrf2-dependent airway epithelial adaptive response to proinflammatory oxidant-hypochlorous acid challenge by transcription profiling
Authors: Zhu Lingxiang , Pi Jingbo , Wachi Shinichiro , Andersen Melvin E. , Wu Reen , Chen Yin
Published: December 2007

In inflammatory diseases of the airway, a high level (estimated to be as high as 8 mM) of HOCl can be generated through a reaction catalyzed by the leukocyte granule enzyme myeloperoxidase (MPO). HOCl, a potent oxidative agent, causes extensive tissue injury through its reaction with various cellular substances, including thiols, nucleotides, and amines. In addition to its physiological source, HOCl can also be generated by chlorine gas inhalation from an accident or a potential terrorist attack. Despite the important role of HOCl-induced airway epithelial injury, the underlying molecular mechanism is largely unknown. In the present study, we found that HOCl induced dose-dependent toxicity in airway epithelial cells. By transcription profiling using GeneChip, we identified a battery of HOCl-inducible antioxidant genes, all of which have been reported previously to be regulated by nuclear factor erythroid-related factor 2 (Nrf2), a transcription factor that is critical to the lung antioxidant response. Consistent with this finding, Nrf2 was found to be activated time and dose dependently by HOCl. Although the epidermal growth factor receptor-MAPK pathway was also highly activated by HOCl, it was not involved in Nrf2 activation and Nrf2-dependent gene expression. Instead, HOCl-induced cellular oxidative stress appeared to lead directly to Nrf2 activation. To further understand the functional significance of Nrf2 activation, small interference RNA was used to knock down Nrf2 level by targeting Nrf2 or enhance nuclear accumulation of Nrf2 by targeting its endogenous inhibitor Keap1. By both methods, we conclude that Nrf2 directly protects airway epithelial cells from HOCl-induced toxicity.
Inflammation and ischemia-induced lung angiogenesis
Authors: Wagner Elizabeth M. , Sánchez Jesús , McClintock Jessica Y. , Jenkins John , Moldobaeva Aigul
Published: December 2007

A role for inflammation in modulating the extent of angiogenesis has been shown for a number of organs. The present study was undertaken to evaluate the importance of leukocyte subpopulations for systemic angiogenesis of the lung after left pulmonary artery ligation (LPAL) in a mouse model of chronic pulmonary thromboembolism. Since we ( 24 ) previously showed that depletion of neutrophils did not alter the angiogenic outcome, we focused on the effects of dexamethasone pretreatment (general anti-inflammatory) and gadolinium chloride treatment (macrophage inactivator) and studied Rag-1−/− mice (T/B lymphocyte deficient). We measured inflammatory cells in bronchoalveolar lavage fluid and lung homogenate macrophage inflammatory protein-2 (MIP-2) and IL-6 protein levels within 24 h after LPAL and systemic blood flow to the lung 14 days after LPAL with labeled microspheres as a measure of angiogenesis. Blood flow to the left lung was significantly reduced after dexamethasone treatment compared with untreated control LPAL mice (66% decrease; P < 0.05) and significantly increased in T/B lymphocyte-deficient mice (88% increase; P < 0.05). Adoptive transfer of splenocytes (T/B lymphocytes) significantly reversed the degree of angiogenesis observed in the Rag-1−/− mice back to the level of control LPAL. Average number of lavaged macrophages for each group significantly correlated with average blood flow in the study groups ( r2 = 0.9181; P = 0.01 different from 0). Despite differences in angiogenesis, left lung homogenate MIP-2 and IL-6 did not differ among study groups. We conclude that inflammatory cells modulate the degree of angiogenesis in this lung model where lymphocytes appear to limit the degree of neovascularization, whereas monocytes/macrophages likely promote angiogenesis.
Self-Reported Delinquency of High School Students in Metro Manila
Authors: Gutierrez Filomin C. , Shoemaker Donald J.
Published: December 2007

Self-report data were gathered from 633 students from public and private schools in metro Manila, Philippines. The study finds overall delinquency prevalence to be higher among males than females but not significantly different from one socioeconomic class to another. Gender and class differentials, however, are found for different types of delinquency (overt property, covert property, theft, swindling, vandalism, drug abuse, alcohol and cigarettes, and status offenses). Violent offenses and more public forms of delinquency are found to be high among lower-class boys, whereas covert types of delinquency are high among the middle- and upper-class students. Of interest, among females, upper-class girls consistently have the highest self-reported delinquency rates.
P‐glycoprotein: So Many Ways to Turn It On
Authors: Callaghan Richard , Crowley Emily , Biochem M , Potter Simon , Kerr Ian D.
Published: December 2007

Expression of the ABC transporter P‐glycoprotein (P‐gp or ABCB1) is associated with resistance to chemotherapy in cancer. However, early investigations into the regulation of ABCB1 expression revealed that the process is not a classical induction as observed for certain metabolizing enzymes. The process involves the cellular stress response pathway initiated by either inflicted (eg, chemotherapy damage) or endogenous (eg, hypoxia) factors. However, ABCB1 is also expressed in a number of noncancerous tissues. In particular, the protein is found at tissues providing a barrier or secretory function. The localization of ABCB1 in normal tissues will impact significantly on drug pharmacokinetics, in particular the absorption and elimination processes. This review also describes the mechanism underlying ABCB1 expression in noncancerous tissue, a process that does not involve the stress response.
Doing Unto Future Selves As You Would Do Unto Others: Psychological Distance and Decision Making
Authors: Pronin Emily , Olivola Christopher Y. , Kennedy Kathleen A.
Published: December 2007

Four experiments showed that the decisions people make for future selves and other people are similar to each other and different from their decisions for present selves. Experiments involved decisions to drink a disgusting liquid for scientific purposes (Experiment 1), tutor peers during exam week (Experiment 2), receive e-mails for charity (Experiment 3), and defer a lottery prize for a larger one (Experiment 4). These findings seemed to be at least partially rooted in the tendency for decisions regarding the ongoing, present self to be uniquely influenced by internal subjective experience. Specifically, these effects emerged for real, but not hypothetical, decisions. Also, they were mitigated by manipulations that altered participants' attention to present or future subjective experience. In addition, when participants' subjective experience primarily involved empathy for others (Experiment 3), their decisions on behalf of present selves were more generous than their decisions for future selves and others. Applications are discussed.
Provider Selection of Evidence-Based Contraception Guidelines in Service Provision
Authors: León Federico R. , Lundgren Rebecka , Jennings Victoria
Published: December 2007

Providers underutilize evidence-based practice guidelines as they prescribe contraceptives. To discern biases in guideline utilization by 172 providers of three countries, this study used observations from simulated clients trained to choose oral contraceptives. Providers implemented less than one third of the guideline set, but they addressed, more frequently than other guidelines, items categorized as essential by expert opinion ( p < .01). Indian providers emphasized instructions on method use in 9-minute consultations, Rwandan providers emphasized contraindications in 29-minute sessions, and Peruvian providers did not emphasize any single guideline category. Providers should use job aids to improve guideline utilization. Those pressed for time need an evidence-based, rather than arbitrary, selection of essential guidelines that optimizes client outcomes. Practice-based research must be generated to meet this need.
Evaluating the Role of Postconstruction Support in Sustaining Drinking Water Projects Evidence from Peru
Authors: Prokopy Linda Stalker , Thorsten Rich , Bakalian Alex , Wakeman Wendy
Published: December 2007

This article assesses the impact of postconstruction support (PCS) on the sustainability of participatory, demand-driven rural water projects in the Cuzco region of Peru. This study evaluates ninety-nine villages from two water supply schemes— projects built under a social investment fund program and those built under a nongovernmental program funded by the Swiss government. Overall, the study finds that the projects are performing very well. Multivariate regression analysis suggests that household- and village-level PCS is linked with financial performance, overall household satisfaction, and attitudes toward long-term future performance after controlling for project and community determinants of sustainability.