bluuebunny/crossref_metadata_2025 huggingface dataset:
Processivity, Substrate Binding, and Mechanism of Cellulose Hydrolysis by
Thermobifida fusca
Cel9A
Authors: Li Yongchao , Irwin Diana C. , Wilson David B.
Published: March 2007

Thermobifida fusca
Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual
T. fusca
enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in
Escherichia coli
; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(−1) O1. R378, a residue interacting with Glc(+1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(−2) to Glc(−4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(−2) to Glc(−4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate.
Targeted Access to the Genomes of Low-Abundance Organisms in Complex Microbial Communities
Authors: Podar Mircea , Abulencia Carl B. , Walcher Marion , Hutchison Don , Zengler Karsten , Garcia Joseph A. , Holland Trevin , Cotton David , Hauser Loren , Keller Martin
Published: March 2007

Current metagenomic approaches to the study of complex microbial consortia provide a glimpse into the community metabolism and occasionally allow genomic assemblies for the most abundant organisms. However, little information is gained for the members of the community present at low frequencies, especially those representing yet-uncultured taxa, which include the bulk of the diversity present in most environments. Here we used phylogenetically directed cell separation by fluorescence in situ hybridization and flow cytometry, followed by amplification and sequencing of a fraction of the genomic DNA of several bacterial cells that belong to the TM7 phylum. Partial genomic assembly allowed, for the first time, a look into the evolution and potential metabolism of a soil representative from this group of organisms for which there are no species in stable laboratory cultures. Genomic reconstruction from targeted cells of uncultured organisms isolated directly from the environment represents a powerful approach to access any specific members of a community and an alternative way to assess the community's metabolic potential.
Control of Ammonium Permease Expression and Filamentous Growth by the GATA Transcription Factors
GLN3
and
GAT1
in
Candida albicans
Authors: Dabas Neelam , Morschhäuser Joachim
Published: March 2007

In response to nitrogen starvation, the human fungal pathogen
Candida albicans
switches from yeast to filamentous growth. This morphogenetic switch is controlled by the ammonium permease Mep2p, whose expression is induced under limiting nitrogen conditions. In order to understand in more detail how nitrogen starvation-induced filamentous growth is regulated in
C. albicans
, we identified the
cis
-acting sequences in the
MEP2
promoter that mediate its induction in response to nitrogen limitation. We found that two putative binding sites for GATA transcription factors have a central role in
MEP2
expression, as deletion of the region containing these sites or mutation of the GATAA sequences in the full-length
MEP2
promoter strongly reduced
MEP2
expression. To investigate whether the GATA transcription factors
GLN3
and
GAT1
regulate
MEP2
expression, we constructed mutants of the
C. albicans
wild-type strain SC5314 lacking one or both of these transcription factors. Expression of Mep2p was strongly reduced in
gln3
Δ and
gat1
Δ single mutants and abolished in
gln3
Δ
gat1
Δ double mutants. Deletion of
GLN3
strongly inhibited filamentous growth under limiting nitrogen conditions, but the filamentation defect of
gln3
Δ mutants could be rescued by constitutive expression of
MEP2
from the
ADH1
promoter. In contrast, inactivation of
GAT1
had no effect on filamentation, and we found that filamentation became independent of the presence of a functional
MEP2
gene in the
gat1
Δ mutants, indicating that the loss of
GAT1
function results in the activation of other pathways inducing filamentous growth. These results demonstrate that the GATA transcription factors
GLN3
and
GAT1
control expression of the
MEP2
ammonium permease and that
GLN3
is also an important regulator of nitrogen starvation-induced filamentous growth in
C. albicans
.
The SsrA-SmpB Ribosome Rescue System Is Important for Growth ofBacillus subtilisat Low and High Temperatures
Authors: Shin Ji-Hyun , Price Chester W.
Published: March 2007

Bacillus subtilishas multiple stress response systems whose integrated action promotes growth and survival under unfavorable conditions. Here we address the function and transcriptional organization of a five-gene cluster containingssrA, previously known to be important for growth at high temperature because of the role of its tmRNA product in rescuing stalled ribosomes. Reverse transcription-PCR experiments detected a single message for thesecG-yvaK-rnr-smpB-ssrAcluster, suggesting that it constitutes an operon. However, rapid amplification of cDNA ends-PCR andlacZfusion experiments indicated that operon transcription is complex, with at least five promoters controlling different segments of the cluster. One σA-like promoter precededsecG(P1), and internal σA-like promoters were found in both thernr-smpB(P2) andsmpB-ssrAintervals (P3and PHS). Another internal promoter lay in thesecG-yvaKintercistronic region, and this activity (PB) was dependent on the general stress factor σB. Null mutations in the four genes downstream from PBwere tested for their effects on growth. Loss ofyvaK(carboxylesterase E) orrnr(RNase R) caused no obvious phenotype. By contrast,smpBwas required for growth at high temperature (52°C), as anticipated if its product (a small ribosomal binding protein) is essential for tmRNA (ssrA) function. Notably,smpBandssrAwere also required for growth at low temperature (16°C), a phenotype not previously associated with tmRNA activity. These results extend the known high-temperature role ofssrAand indicate that the ribosome rescue system is important at both extremes of theB. subtilistemperature range.
Identification of the
l
,
d
-Transpeptidases Responsible for Attachment of the Braun Lipoprotein to
Escherichia coli
Peptidoglycan
Authors: Magnet Sophie , Bellais Samuel , Dubost Lionel , Fourgeaud Martine , Mainardi Jean-Luc , Petit-Frère Sébastien , Marie Arul , Mengin-Lecreulx Dominique , Arthur Michel , Gutmann Laurent
Published: March 2007

The
l
,
d
-transpeptidase Ldt
fm
catalyzes peptidoglycan cross-linking in β-lactam-resistant mutant strains of
Enterococcus faecium
. Here, we show that in
Escherichia coli
Ldt
fm
homologues are responsible for the attachment of the Braun lipoprotein to murein, indicating that evolutionarily related domains have been tailored to use muropeptides or proteins as acyl acceptors in the
l
,
d
-transpeptidation reaction.
Septum-Localized Protein Required for Filament Integrity and Diazotrophy in the Heterocyst-Forming Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Flores Enrique , Pernil Rafael , Muro-Pastor Alicia M. , Mariscal Vicente , Maldener Iris , Lechno-Yossef Sigal , Fan Qing , Wolk C. Peter , Herrero Antonia
Published: March 2007

Heterocysts, formed when filamentous cyanobacteria, such as
Anabaena
sp. strain PCC 7120, are grown in the absence of combined nitrogen, are cells that are specialized in fixing atmospheric nitrogen (N
2
) under oxic conditions and that transfer fixed nitrogen to the vegetative cells of the filament.
Anabaena
sp. mutants whose
sepJ
gene (open reading frame
alr2338
of the
Anabaena
sp. genome) was affected showed filament fragmentation and arrested heterocyst differentiation at an early stage. In a
sepJ
insertional mutant, a layer similar to a heterocyst polysaccharide layer was formed, but the heterocyst-specific glycolipids were not synthesized. The
sepJ
mutant did not exhibit nitrogenase activity even when assayed under anoxic conditions. In contrast to proheterocysts produced in the wild type, those produced in the
sepJ
mutant still divided. SepJ is a multidomain protein whose N-terminal region is predicted to be periplasmic and whose C-terminal domain resembles an export permease. Using a green fluorescent protein translationally fused to the carboxyl terminus of SepJ, we observed that in mature heterocysts and vegetative cells, the protein is localized at the intercellular septa, and when cell division starts, it is localized in a ring whose position is similar to that of a Z ring. SepJ is a novel composite protein needed for filament integrity, proper heterocyst development, and diazotrophic growth.
Regulation of
dev
, an Operon That Includes Genes Essential for
Myxococcus xanthus
Development and CRISPR-Associated Genes and Repeats
Authors: Viswanathan Poorna , Murphy Kimberly , Julien Bryan , Garza Anthony G. , Kroos Lee
Published: March 2007

Expression of
dev
genes is important for triggering spore differentiation inside
Myxococcus xanthus
fruiting bodies. DNA sequence analysis suggested that
dev
and
cas
(
C
RISPR-
as
sociated) genes are cotranscribed at the
dev
locus, which is adjacent to CRISPR (
c
lustered
r
egularly
i
nterspaced
s
hort
p
alindromic
r
epeats). Analysis of RNA from developing
M. xanthus
confirmed that
dev
and
cas
genes are cotranscribed with a short upstream gene and at least two repeats of the downstream CRISPR, forming the
dev
operon. The operon is subject to strong, negative autoregulation during development by DevS. The
dev
promoter was identified. Its −35 and −10 regions resemble those recognized by
M. xanthus
σ
A
RNA polymerase, the homolog of
Escherichia coli
σ
70
, but the spacer may be too long (20 bp); there is very little expression during growth. Induction during development relies on at least two positive regulatory elements located in the coding region of the next gene upstream. At least two positive regulatory elements and one negative element lie downstream of the
dev
promoter, such that the region controlling
dev
expression spans more than 1 kb. The results of testing different fragments for
dev
promoter activity in wild-type and
devS
mutant backgrounds strongly suggest that upstream and downstream regulatory elements interact functionally. Strikingly, the 37-bp sequence between the two CRISPR repeats that, minimally, are cotranscribed with
dev
and
cas
genes exactly matches a sequence in the bacteriophage Mx8
intP
gene, which encodes a form of the integrase needed for lysogenization of
M. xanthus
.
Transcription Activation Mediated by a Cyclic AMP Receptor Protein from
Thermus thermophilus
HB8
Authors: Shinkai Akeo , Kira Satoshi , Nakagawa Noriko , Kashihara Aiko , Kuramitsu Seiki , Yokoyama Shigeyuki
Published: March 2007

The extremely thermophilic bacterium
Thermus thermophilus
HB8, which belongs to the phylum
Deinococcus
-
Thermus
, has an open reading frame encoding a protein belonging to the cyclic AMP (cAMP) receptor protein (CRP) family present in many bacteria. The protein named
T. thermophilus
CRP is highly homologous to the CRP family proteins from the phyla
Firmicutes
,
Actinobacteria
, and
Cyanobacteria
, and it forms a homodimer and interacts with cAMP. CRP mRNA and intracellular cAMP were detected in this strain, which did not drastically fluctuate during cultivation in a rich medium. The expression of several genes was altered upon disruption of the
T. thermophilus
CRP gene. We found six CRP-cAMP-dependent promoters in in vitro transcription assays involving DNA fragments containing the upstream regions of the genes exhibiting decreased expression in the CRP disruptant, indicating that the CRP is a transcriptional activator. The consensus
T. thermophilus
CRP-binding site predicted upon nucleotide sequence alignment is 5′-(C/T)NNG(G/T)(G/T)C(A/C)N(A/T)NNTCACAN(G/C)(G/C)-3′. This sequence is unique compared with the known consensus binding sequences of CRP family proteins. A putative −10 hexamer sequence resides at 18 to 19 bp downstream of the predicted
T. thermophilus
CRP-binding site. The CRP-regulated genes found in this study comprise clustered regularly interspaced short palindromic repeat (CRISPR)-associated (
cas
) ones, and the genes of a putative transcriptional regulator, a protein containing the exonuclease III-like domain of DNA polymerase, a GCN5-related acetyltransferase homolog, and
T. thermophilus
-specific proteins of unknown function. These results suggest a role for cAMP signal transduction in
T. thermophilus
and imply the
T. thermophilus
CRP is a cAMP-responsive regulator.
Identification and Characterization of Genes Encoding a Putative ABC-Type Transporter Essential for Utilization of γ-Hexachlorocyclohexane in
Sphingobium japonicum
UT26
Authors: Endo Ryo , Ohtsubo Yoshiyuki , Tsuda Masataka , Nagata Yuji
Published: March 2007

Sphingobium japonicum
UT26 utilizes γ-hexachlorocyclohexane (γ-HCH) as its sole source of carbon and energy. In our previous studies, we cloned and characterized genes encoding enzymes for the conversion of γ-HCH to β-ketoadipate in UT26. In this study, we analyzed a mutant obtained by transposon mutagenesis and identified and characterized new genes encoding a putative ABC-type transporter essential for the utilization of γ-HCH in strain UT26. This putative ABC transporter consists of four components, permease, ATPase, periplasmic protein, and lipoprotein, encoded by
linK
,
linL
,
linM
, and
linN
, respectively. Mutation and complementation analyses indicated that all the
linKLMN
genes are required, probably as a set, for γ-HCH utilization in UT26. Furthermore, the mutant cells deficient in this putative ABC transporter showed (i) higher γ-HCH degradation activity and greater accumulation of the toxic dead-end product 2,5-dichlorophenol (2,5-DCP), (ii) higher sensitivity to 2,5-DCP itself, and (iii) higher permeability of hydrophobic compounds than the wild-type cells. These results strongly suggested that LinKLMN are involved in γ-HCH utilization by controlling membrane hydrophobicity. This study clearly demonstrated that a cellular factor besides catabolic enzymes and transcriptional regulators is essential for utilization of xenobiotic compounds in bacterial cells.
Nodal aggressive B-cell lymphomas: a diagnostic approach
Authors: Prakash Sonam , Swerdlow Steven H
Published: March 2007

The diffuse aggressive B-cell lymphomas, as recognised in the 2001 WHO classification, represent a clinically and biologically heterogeneous group of neoplasms that require very different therapeutic approaches and have very different outcomes. They should be diagnosed using a multiparameter approach that emphasises morphological and immunophenotypic studies, and in at least some cases, relies on cytogenetic and/or genotypic studies. Incorporation of clinical data may be important as well. There is also current interest in going beyond the basic diagnosis and providing pathological prognostic information when possible. Whereas the diagnosis of some cases will be relatively easy, the differential diagnosis in others is very difficult, with some cases in a grey zone between two different well defined categories.
Intraoperative assessment of the Whipple resection specimen
Authors: Khalifa Mahmoud A
Published: March 2007

Intraoperative consultation for the Whipple resection procedure has evolved due to the increasing influence of imaging techniques in surgical planning and decision-making. The indications and utilisation of this service vary, at least to some degree, from one institution to the other. The following discussion is a single institutional approach, which is hoped to provide assistance to the practising pathologists in this field. Special emphasis is given to the relevant anatomical considerations and the most common indications for an intraoperative consultation.
Complex patterns of chromosome 9 alterations including the p16INK4alocus in Wilms tumours
Authors: Natrajan R , Warren W , Messahel B , Reis-Filho J S , Brundler M-A , Dome J S , Grundy P E , Vujanic G , Pritchard-Jones K , Jones C
Published: March 2007

Background:

Previous data implicating genetic and epigenetic events on chromosome 9, including theCDKN2A/2Blocus, as molecular predictors of Wilms tumour relapse, have been conflicting.

Aims:

To clarify this using genome-wide and focused molecular genetic analysis.

Methods:

Microarray-based comparative genomic hybridisation (aCGH) using genome-wide coverage was applied to 76 favourable histology Wilms tumours. Additional investigation of the 9p21 locus was carried out using loss of heterozygosity (LOH) and fluorescence in situ hybridisation (FISH), as well as immunohistochemistry for CDKN2A/p16INK4aon a paediatric renal tumour tissue microarray.

Results:

Approximately half of the tumours were found to show chromosome 9 copy number changes. Those cases which harboured alterations comprised at least four distinct patterns: gain of the entire chromosome, loss of 9p, gain of 9q34, or a more complex combination of gains/losses. None of these tumour groups showed any statistically significant correlation with clinicopathological variables. Deletion mapping of 9p by LOH revealed several regions of overlap, including theCDKN2A/2Blocus in 4/34 (11.8%) tumours, which was confirmed to represent hemizygous deletions by FISH. CDKN2A/p16INK4aprotein expression was predominantly negative in Wilms tumours as assessed by immunohistochemistry on a tissue array, reflecting the expression pattern in normal kidney. However, 38/236 (16.1%) non-anaplastic Wilms tumours, 4/9 (44.4%) anaplastic Wilms tumours, 5/7 (71.4%) rhabdoid tumours of the kidney, and 4/10 (40%) clear cell sarcomas of the kidney showed nuclear CDKN2A/p16INK4aimmunoreactivity.

Conclusions:

These data reveal the complex nature of genetic alterations on chromosome 9 in Wilms tumours, but do not provide evidence for their involvement in or association with treatment failure.
First Responders: Mental Health Consequences of Natural and Human-Made Disasters for Public Health and Public Safety Workers
Authors: Benedek David M. , Fullerton Carol , Ursano Robert J.
Published: March 2007

First responders, including military health care workers, public health service workers, and state, local, and volunteer first responders serve an important role in protecting our nation's citizenry in the aftermath of disaster. Protecting our nation's health is a vital part of preserving national security and the continuity of critical national functions. However, public health and public safety workers experience a broad range of health and mental health consequences as a result of work-related exposures to natural or man-made disasters. This chapter reviews recent epidemiologic studies that broaden our understanding of the range of health and mental health consequences for first responders. Evidence-based psychopharmacologic and psychotherapeutic interventions for posttraumatic distress reactions and psychiatric disorders are outlined. Finally, the application of public health intervention models for the assessment and management of distress responses and mental disorders in first-responder communities is discussed.
Adverse Late Effects of Childhood Cancer and Its Treatment on Health and Performance
Authors: Ness Kirsten K. , Gurney James G.
Published: March 2007

More than 12,000 newly diagnosed cases of cancer occur each year in the United States among children ages 20 years or younger, and the current 5-year survival rate is near 80%. An estimated 228,000 among adults 47 years or younger and currently living in the United States had a diagnosis of cancer during childhood or adolescence. Here, we review long-term adverse effects of childhood cancer and its treatment with an emphasis on physical performance and health. We also briefly review existing guidelines that may be used to develop appropriate exercise and diet interventions for childhood cancer survivors. We suggest that there is a need for development of evidence-based, risk-based guidelines and interventions for health promotion among long-term childhood cancer survivors, particularly for those whose physical activity limitations interfere with chances for optimizing their bodies' potential in today's society.
Youth Violence Prevention Comes of Age: Research, Training and Future Directions
Authors: Williams Kara , Rivera Lourdes , Neighbours Robert , Reznik Vivian
Published: March 2007

Youth violence is recognized as a major public health problem in the United States and the world. Over the past ten years, progress has been made in documenting the factors that contribute to violent behavior. Emerging research is deepening our understanding of the individual and societal influences that contribute to and protect against youth violence. However, much work still remains to be done in this field, both in examining potential causes and in designing effective intervention strategies. This chapter highlights specific dimensions of youth violence prevention selected by the authors because these dimensions are the focus of public attention, are emerging as critical issues in the study of youth violence, or have a unique place in the current political and social context. We focus on the developmental pathways to violence, factors that mediate and moderate youth violence, the role of culture and media in youth violence, school-based violence such as school shootings and bullying, and the training of health care professionals.
Network Analysis in Public Health: History, Methods, and Applications
Authors: Luke Douglas A. , Harris Jenine K.
Published: March 2007

Network analysis is an approach to research that is uniquely suited to describing, exploring, and understanding structural and relational aspects of health. It is both a methodological tool and a theoretical paradigm that allows us to pose and answer important ecological questions in public health. In this review we trace the history of network analysis, provide a methodological overview of network techniques, and discuss where and how network analysis has been used in public health. We show how network analysis has its roots in mathematics, statistics, sociology, anthropology, psychology, biology, physics, and computer science. In public health, network analysis has been used to study primarily disease transmission, especially for HIV/AIDS and other sexually transmitted diseases; information transmission, particularly for diffusion of innovations; the role of social support and social capital; the influence of personal and social networks on health behavior; and the interorganizational structure of health systems. We conclude with future directions for network analysis in public health.
Rationale and Public Health Implications of Changing CHD Risk Factor Definitions
Authors: Kaplan Robert M. , Ong Michael
Published: March 2007

The definition of disease is central to the practice of medicine and to public health policy. Practice guidelines set standards for disease identification and treatment. Quality care is often defined as adherence to these guidelines. Over the past few years, the diagnostic thresholds for several common medical conditions have been lowered, resulting in a substantial expansion in the market for health care. The most recent guidelines for high blood pressure, high cholesterol, and impaired fasting glucose each define a high percentage of the adult population as in need of regular medical attention. Under the latest proposed thresholds, virtually the entire adult population qualifies for a chronic condition diagnosis. We evaluate the health and financial outcomes associated with changes in diagnostic thresholds for the prevention of three risk factors for cardiovascular disease and stroke: blood pressure, serum cholesterol, and fasting plasma glucose. Estimates of the numbers of people affected, the cost implications, and the overall public health consequences are offered.
Role of calcitonin receptor-like receptor in colonic motility and inflammation
Authors: Clifton Matthew S. , Hoy Julia J. , Chang Jen , Idumalla Prema S. , Fakhruddin Humera , Grady Eileen F. , Dada Stephen , Corvera Carlos U. , Bhargava Aditi
Published: March 2007

Calcitonin gene-related peptide (CGRP) mediates neurogenic inflammation and modulates intestinal motility. The CGRP receptor is a heterodimer of calcitonin receptor-like receptor (CLR) and receptor-associated modifying protein 1. We used RNA interference to elucidate the specific role of CLR in colonic motility and inflammation. Intramural injection of double-stranded RNA (dsRNA) against CLR (dsCLR) into the colonic wall at two sites caused the spatial and temporal downregulation of CLR in the colon within 1 day of dsRNA injection. Knockdown of CLR persisted for 7–9 days, and the effect of knockdown spread to ∼2 cm proximal and distal to the injection sites, whereas control dsRNA injection did not affect CLR expression. Measurement of isometric contractions of isolated colonic muscle segments revealed that in control dsRNA-injected rats, CGRP abrogated contractions entirely and decreased resting muscular tone, whereas in dsCLR-injected rats, CGRP decreased muscle tone but slow-wave contractions of varying amplitude persisted. In trinitrobenzene sulfonic acid-induced colitis, rats with knockdown of CLR displayed a significantly greater degree of edema and necrosis than saline- or control dsRNA-injected rats. Levels of the proinflammatory cytokines TNF-α and IL-6 were markedly upregulated by trinitrobenzene sulfonic acid treatment. TNF-α mRNA levels were further increased in CLR knockdown rats, whereas levels of IL-6 were unaltered. Thus this study demonstrates that CLR is a functional receptor for CGRP.
Metformin and phenformin activate AMP-activated protein kinase in the heart by increasing cytosolic AMP concentration
Authors: Zhang Li , He Huamei , Balschi James A.
Published: March 2007

AMP-activated protein kinase (AMPK) acts as a cellular energy sensor: it responds to an increase in AMP concentration ([AMP]) or the AMP-to-ATP ratio (AMP/ATP). Metformin and phenformin, which are biguanides, have been reported to increase AMPK activity without increasing AMP/ATP. This study tests the hypothesis that these biguanides increase AMPK activity in the heart by increasing cytosolic [AMP]. Groups of isolated rat hearts ( n = 5–7 each) were perfused with Krebs-Henseleit buffer with or without 0.2 mM phenformin or 10 mM metformin, and31P-NMR-measured phosphocreatine, ATP, and intracellular pH were used to calculate cytosolic [AMP]. At various times, hearts were freeze-clamped and assayed for AMPK activity, phosphorylation of Thr172on AMPK-α, and phosphorylation of Ser79on acetyl-CoA carboxylase, an AMPK target. In hearts treated with phenformin for 18 min and then perfused for 20 min with Krebs-Henseleit buffer, [AMP] began to increase at 26 min and AMPK activity was elevated at 36 min. In hearts treated with metformin, [AMP] was increased at 50 min and AMPK activity, phosphorylated AMPK, and phosphorylated acetyl-CoA carboxylase were elevated at 61 min. In metformin-treated hearts, HPLC-measured total AMP content and total AMP/ATP did not increase. In summary, phenformin and metformin increase AMPK activity and phosphorylation in the isolated heart. The increase in AMPK activity was always preceded by and correlated with increased cytosolic [AMP]. Total AMP content and total AMP/ATP did not change. Cytosolic [AMP] reported metabolically active AMP, which triggered increased AMPK activity, but measures of total AMP did not.
Myocardial hypoxia-inducible HIF-1α, VEGF, and GLUT1 gene expression is associated with microvascular and ICAM-1 heterogeneity during endotoxemia
Authors: Bateman Ryon M. , Tokunaga Chiho , Kareco Thoma , Dorscheid Delbert R. , Walley Keith R.
Published: March 2007

The systemic inflammatory response to infection is the leading cause of mortality in North American intensive-care units. Although much is known about inflammatory mediators, the relationships between microregional inflammation, microvascular heterogeneity, hypoxia, hypoxia-inducible gene expression, and myocardial dysfunction are unknown. Male Sprague-Dawley rats were injected intraperitoneally with LPS to test the hypothesis that sepsis-induced local inflammation and increased microvascular heterogeneity are spatially and temporally associated with hypoxia, hypoxia-inducible gene expression, and decreased left-ventricular contractility. Using a combination of three-dimensional microvascular imaging, tissue Po2, and pressure-volume conductance measurements, we found that 5 h after LPS, minimum oxygen-diffusion distances increased ( P < 0.05), whereas tissue oxygenation and contractility both decreased ( P < 0.05) in the left ventricle. Real-time RT-PCR analysis revealed that the hypoxia-inducible genes hypoxia-inducible factor (HIF)-1α, VEGF, and glucose transporter (GLUT) 1 were all upregulated ( P < 0.05) in the left ventricle. Tissue regions expressing ICAM-1, obtained by using laser-capture microdissection, had increased HIF-1α and GLUT1 ( P < 0.05) gene expression. VEGF gene expression was more diffuse. In LPS rats, GLUT1 gene expression correlated ( P < 0.05) with left-ventricular contractility. In 5-h hypoxic cardiomyocytes, we found strong transient HIF-1α, weak VEGF, and greater prolonged GLUT1 gene expression. By comparison, the HIF-1α-GLUT1 gene-induction pattern was reversed in the left ventricle of LPS rats. Together, these results show that LPS induces hypoxia in the left ventricle associated with increased microvascular heterogeneity and decreased contractility. HIF-1α and GLUT1 gene induction are related to a heterogeneous ICAM-1 expression and may be cardioprotective during the onset of septic injury.
Role of oxidative stress in cardiac dysfunction of PPARα−/−mice
Authors: Guellich Aziz , Damy Thibaud , Lecarpentier Yves , Conti Marc , Claes Victor , Samuel Jane-Lyse , Quillard Jeanine , Hébert Jean-Louis , Pineau Thierry , Coirault Catherine
Published: March 2007

This study was designed to determine the effects of PPARα lack on cardiac mechanical performance and to identify potential intracellular mechanisms linking PPARα pathway deficiency to cardiac contractile dysfunction. Echocardiography, ex vivo papillary muscle assays, and in vitro motility assays were used to assess global, intrinsic ventricular muscle performance and myosin mechanical properties, respectively, in PPARα−/−and age-matched wild-type mice. Three-nitrotyrosine formation and 4-hydroxy-2-nonenal protein-adducts, both markers of oxidative damage, were analyzed by Western blot analysis and immunolabeling. Radical scavenging capacity was analyzed by measuring protein levels and/or activities of the main antioxidant enzymes, including catalase, glutathione peroxidase, and manganese and copper-zinc superoxide dismutases. Echocardiographic left ventricular fractional shortening in PPARα−/−was 16% lower than that in wild-type. Ex vivo left ventricular papillary muscle exhibited reduced shortening velocity and isometric tension (three- and twofold, respectively). In vitro myosin-based velocity was ≈20% slower in PPARα−/−, indicating that myosin itself was involved in the contractile dysfunction. Staining of 3-nitrotyrosine was more pronounced in PPARα−/−, and myosin heavy chain was the main nitrated protein. Formation of 3-nitrotyrosine myosin heavy chain was twofold higher in PPARα−/−and 4-hydroxy-2-nonenal protein-adducts were threefold higher. The expression and activity of manganese superoxide dismutase were respectively 33% and 50% lower in PPARα−/−, with no changes in copper-zinc superoxide dismutase, catalase, or glutathione peroxidase. These findings demonstrate that PPARα pathway deficiency impairs cardiac function and also identify oxidative damage to myosin as a link between PPARα deficiency and contractile dysfunction.
Effects of hyperglycemia on the cerebrovascular response to rhythmic handgrip exercise
Authors: Kim Yu-Sok , Krogh-Madsen Rikke , Rasmussen Peter , Plomgaard Peter , Ogoh Shigehiko , Secher Niels H. , van Lieshout Johannes J.
Published: March 2007

Dynamic cerebral autoregulation (CA) is challenged by exercise and may become less effective when exercise is exhaustive. Exercise may increase arterial glucose concentration, and we evaluated whether the cerebrovascular response to exercise is affected by hyperglycemia. The effects of a hyperinsulinemic euglycemic clamp (EU) and hyperglycemic clamp (HY) on the cerebrovascular (CVRI) and systemic vascular resistance index (SVRI) responses were evaluated in seven healthy subjects at rest and during rhythmic handgrip exercise. Transfer function analysis of the dynamic relationship between beat-to-beat changes in mean arterial pressure and middle cerebral artery (MCA) mean blood flow velocity ( Vmean) was used to assess dynamic CA. At rest, SVRI decreased with HY and EU ( P < 0.01). CVRI was maintained with EU but became reduced with HY [11% (SD 3); P < 0.01], and MCA Vmean increased ( P < 0.05), whereas brain catecholamine uptake and arterial Pco2 did not change significantly. HY did not affect the normalized low-frequency gain between mean arterial pressure and MCA Vmean or the phase shift, indicating maintained dynamic CA. With HY, the increase in CVRI associated with exercise was enhanced (19 ± 7% vs. 9 ± 7%; P < 0.05), concomitant with a larger increase in heart rate and cardiac output and a larger reduction in SVRI (22 ± 4% vs. 14 ± 2%; P < 0.05). Thus hyperglycemia lowered cerebral vascular tone independently of CA capacity at rest, whereas dynamic CA remained able to modulate cerebral blood flow around the exercise-induced increase in MCA Vmean. These findings suggest that elevated blood glucose does not explain that dynamic CA is affected during intense exercise.
Disturbance of macro- and microcirculation: relations with pulse pressure and cardiac organ damage
Authors: Safar Michel E. , Lacolley P.
Published: March 2007

Whereas large arteries dampen oscillations resulting from intermittent ventricular ejection, small arteries steadily deliver optimal blood flow to various organs as the heart. The transition from pulsatile to steady pressure is influenced by several factors as wave travel, damping, and reflections, which are mainly determined by the impedance mismatch between large vessels and arteriolar bifurcations. The mechanism(s) behind the dampening of pressure wave in the periphery and the links between central and peripheral pulsatile pressure (PP) may determine cardiac damage. Active pathways participate to pulse widening and changes in pulse amplitude in microvessels. Steady and cyclic stresses operate through different transduction mechanisms, the former being focal adhesion kinase and the latter being free radicals and oxidative stress. Independently of mechanics, calcifications and attachment molecules contribute to enhance vessel wall stiffness through changes in collagen cross-links, proteoglycans, integrins, and fibronectin. Enhanced PP transmission may thus occur and precipitate organ damage at each time that autoregulatory mechanisms, normally protecting the heart from vascular injury, are blunted. Such circumstances, observed in old subjects with systolic hypertension and/or Type 2 diabetes mellitus, particularly under high-sodium diet, cause cardiac damage and explain why increased PP and arterial stiffness are significant predictors of morbidity and mortality in the elderly.
Activation of a cGMP-sensitive calcium-dependent chloride channel may cause transition from calcium waves to whole cell oscillations in smooth muscle cells
Authors: Jacobsen Jens Christian Brings , Aalkjær Christian , Nilsson Holger , Matchkov Vladimir V. , Freiberg Jacob , Holstein-Rathlou Niels-Henrik
Published: March 2007

In vitro, α-adrenoreceptor stimulation of rat mesenteric small arteries often leads to a rhythmic change in wall tension, i.e., vasomotion. Within the individual smooth muscle cells of the vascular wall, vasomotion is often preceded by a period of asynchronous calcium waves. Abruptly, these low-frequency waves may transform into high-frequency whole cell calcium oscillations. Simultaneously, multiple cells synchronize, leading to rhythmic generation of tension. We present a mathematical model of vascular smooth muscle cells that aims at characterizing this sudden transition. Simulations show calcium waves sweeping through the cytoplasm when the sarcoplasmic reticulum (SR) is stimulated to release calcium. A rise in cGMP leads to the experimentally observed transition from waves to whole cell calcium oscillations. At the same time, membrane potential starts to oscillate and the frequency approximately doubles. In this transition, the simulated results point to a key role for a recently discovered cGMP-sensitive calcium-dependent chloride channel. This channel depolarizes the membrane in response to calcium released from the SR. In turn, depolarization causes a uniform opening of L-type calcium channels on the cell surface, stimulating a synchronized release of SR calcium and inducing the shift from waves to whole cell oscillations. The effect of the channel is therefore to couple the processes of the SR with those of the membrane. We hypothesize that the shift in oscillatory mode and the associated onset of oscillations in membrane potential within the individual cell may underlie sudden intercellular synchronization and the appearance of vasomotion.
A model of smooth muscle cell synchronization in the arterial wall
Authors: Jacobsen Jens Christian Brings , Aalkjær Christian , Nilsson Holger , Matchkov Vladimir V. , Freiberg Jacob , Holstein-Rathlou Niels-Henrik
Published: March 2007

Vasomotion is a rhythmic variation in microvascular diameter. Although known for more than 150 years, the cellular processes underlying the initiation of vasomotion are not fully understood. In the present study a model of a single cell is extended by coupling a number of cells into a tube. The simulated results point to a permissive role of cGMP in establishing intercellular synchronization. In sufficient concentration, cGMP may activate a cGMP-sensitive calcium-dependent chloride channel, causing a tight spatiotemporal coupling between release of sarcoplasmic reticulum calcium, membrane depolarization, and influx of extracellular calcium. Low [cGMP] is associated only with unsynchronized waves. At intermediate concentrations, cells display either waves or whole cell oscillations, but these remain unsynchronized between cells. Whole cell oscillations are associated with rhythmic variation in membrane potential and flow of current through gap junctions. The amplitude of these oscillations in potential grows with increasing [cGMP], and, past a certain threshold, they become strong enough to entrain all cells in the vascular wall, thereby initiating sustained vasomotion. In this state there is a rhythmic flow of calcium through voltage-sensitive calcium channels into the cytoplasm, making the frequency of established vasomotion sensitive to membrane potential. It is concluded that electrical coupling through gap junctions is likely to be responsible for the rapid synchronization across a large number of cells. Gap-junctional current between cells is due to the appearance of oscillations in the membrane potential that again depends on the entrainment of sarcoplasmic reticulum and plasma membrane within the individual cell.
Effect of simulated postprandial hyperglycemia on coronary blood flow in cardiac transplant recipients
Authors: McNulty Patrick H. , Tulli Mark A. , Robertson Bryan J. , Lendel Vasili , Harach Lisa A. , Scott Sofia , Boehmer John P.
Published: March 2007

Patients with diabetes mellitus exhibit postprandial hyperglycemia, systemic oxidative stress, impaired endothelium-dependent, nitric oxide (NO)-mediated coronary artery dilatation, and an increased incidence of coronary events. Whether hyperglycemia causally mediates these associations is unknown. To test the hypothesis that postprandial hyperglycemia acutely impairs coronary endothelial function in humans, we compared the ability of the endothelium-dependent vasodilator acetylcholine to increase conduit coronary diameter (the macrovascular response) and coronary blood flow velocity (the microvascular response) in 12 cardiac transplant recipients without diabetes before and after blood glucose was raised from 6.7 ± 1.3 mmol/l (121 ± 24 mg/dl) to 17.8 ± 1.5 mmol/l (321 ± 27 mg/dl) for 1 h. Hyperglycemia acutely doubled circulating levels of the oxidation product malondialdehyde, indicating systemic oxidative stress, but did not affect the ability of acetylcholine to dilate conduit coronary segments or accelerate coronary blood flow. We conclude that the oxidative stress associated with a single acute episode of hyperglycemia affects neither acetylcholine-mediated coronary endothelial NO release nor the subsequent bioavailability, metabolism, or action of NO within the coronary circulation of cardiac transplant recipients. These observations imply that the relationship among hyperglycemia, oxidative stress, and coronary endothelial dysfunction is presumably mediated by mechanisms operating over longer periods of time.
Abolition of reperfusion-induced arrhythmias in hearts from thiamine-deficient rats
Authors: Oliveira Fernando A. , Guatimosim Silvia , Castro Carlos H. , Galan Diogo T. , Lauton-Santos Sandra , Ribeiro Angela M. , Almeida Alvair P. , Cruz Jader S.
Published: March 2007

Extensive work has been done regarding the impact of thiamine deprivation on the nervous system. In cardiac tissue, chronic thiamine deficiency is described to cause changes in the myocardium that can be associated with arrhythmias. However, compared with the brain, very little is known about the effects of thiamine deficiency on the heart. Thus this study was undertaken to explore whether thiamine deprivation has a role in cardiac arrhythmogenesis. We examined hearts isolated from thiamine-deprived and control rats. We measured heart rate, diastolic and systolic tension, and contraction and relaxation rates. Whole cell voltage clamp was performed in rat isolated cardiac myocytes to measure L-type Ca2+current. In addition, we investigated the global intracellular calcium transients by using confocal microscopy in the line-scan mode. The hearts from thiamine-deficient rats did not degenerate into ventricular fibrillation during 30 min of reperfusion after 15 min of coronary occlusion. The antiarrhythmogenic effects were characterized by the arrhythmia severity index. Our results suggest that hearts from thiamine-deficient rats did not experience irreversible arrhythmias. There was no change in L-type Ca2+current density. Inactivation kinetics of this current in Ca2+-buffered cells was retarded in thiamine-deficient cardiac myocytes. The global Ca2+release was significantly reduced in thiamine-deficient cardiac myocytes. The amplitude of caffeine-releasable Ca2+was lower in thiamine-deficient myocytes. In summary, we have found that thiamine deprivation attenuates the incidence and severity of postischemic arrhythmias, possibly through a mechanism involving a decrease in global Ca2+release.
Contributory role of VEGF overexpression in endothelin-1-induced cardiomyocyte hypertrophy
Authors: Shimojo Nobutake , Jesmin Subrina , Zaedi Sohel , Otsuki Takeshi , Maeda Seiji , Yamaguchi Naoto , Aonuma Kazutaka , Hattori Yuichi , Miyauchi Takashi
Published: March 2007

Although endothelin-1 (ET-1) stimulates vascular endothelial growth factor (VEGF) expression in a variety of cells, including endothelial cells and vascular smooth muscle cells, the effects of ET-1 on expression of VEGF and its receptors in cardiomyocytes are unknown. In the present study, we found that treatment of neonatal rat cardiomyocytes with ET-1 for 24 h resulted in upregulation of VEGF and its two principal receptors, fetal liver kinase 1 and fms-like tyrosine kinase 1, in a concentration-dependent manner (10−12 to 10−6 M). ET-1 treatment also caused significant cardiomyocyte hypertrophy, as indicated by increases in cell surface area and [14C]leucine uptake by cardiomyocytes. Treatment with TA-0201 (10−6 M), an ETA-selective blocker, eliminated ET-1-induced overexpression of VEGF and its receptors as well as cardiomyocyte hypertrophy. Treatment with VEGF neutralizing peptides (5–10 μg/ml) partially but significantly inhibited ET-1-induced cardiomyocyte hypertrophy. These results suggest that ET-1 treatment of cardiomyocytes promotes overexpression of VEGF and its receptors via activation of ETA receptors, and consequently the upregulated VEGF signaling system appears to contribute, at least in part, to ET-1-induced cardiomyocyte hypertrophy.
Diastolic ventricular interactions in endurance-trained athletes during orthostatic stress
Authors: Esch Ben T. A. , Scott Jessica M. , Haykowsky Mark J. , McKenzie Don C. , Warburton Darren E. R.
Published: March 2007

Enhanced left-ventricular (LV) compliance is a common adaptation to endurance training. This adaptation may have differential effects under conditions of altered venous return. The purpose of this investigation was to assess the effect of cardiac (un)loading on right ventricular (RV) cavity dimensions and LV volumes in endurance-trained athletes and normally active males. Eight endurance-trained (Vo2max, 65.4 ± 5.7 ml·kg−1·min−1) and eight normally active (Vo2max, 45.1 ± 6.0 ml·kg−1·min−1) males underwent assessments of the following: 1) Vo2max, 2) orthostatic tolerance, and 3) cardiac responses to lower-body positive (0–60 mmHg) and negative (0 to −80 mmHg) pressures with echocardiography. In response to negative pressures, echocardiographic analysis revealed a similar decrease in RV end-diastolic cavity area in both groups (e.g., at −80 mmHg: normals, 21.4%; athletes, 20.8%) but a greater decrease in LV end-diastolic volume in endurance-trained athletes (e.g., at −80 mmHg: normals, 32.3%; athletes, 44.4%; P < 0.05). Endurance-trained athletes also had significantly greater decreases in LV stroke volume during lower-body negative pressure. During positive pressures, endurance-trained athletes showed larger increases in LV end-diastolic volume (e.g., at +60 mmHg; normals, 14.1%; athletes, 26.8%) and LV stroke volume, despite similar responses in RV end-diastolic cavity area (e.g., at +60 mmHg: normals, 18.2%; athletes, 24.2%; P < 0.05). This investigation revealed that in response to cardiac (un)loading similar changes in RV cavity area occur in endurance-trained and normally active individuals despite a differential response in the left ventricle. These differences may be the result of alterations in RV influence on the left ventricle and/or intrinsic ventricular compliance.