bluuebunny/crossref_metadata_2025 huggingface dataset:
Interstrain Gene Transfer inChlamydia trachomatisIn Vitro: Mechanism and Significance
Authors: DeMars Robert , Weinfurter Jason
Published: December 2007

The high frequency of between-strain genetic recombinants ofChlamydia trachomatisamong isolates obtained from human sexually transmitted infections suggests that lateral gene transfer (LGT) is an important means by whichC. trachomatisgenerates variants that have enhanced relative fitness. A mechanism for LGT inC. trachomatishas not been described, and investigation of this phenomenon by experimentation has been hampered by the obligate intracellular development of this pathogen. We describe here experiments that readily detected LGT between strains ofC. trachomatisin vitro. Host cells were simultaneously infected with an ofloxacin-resistant (Ofxr) mutant of a serovar L1 strain (L1:Ofxr-1) and a rifampin-resistant (Rifr) mutant of a serovar D strain (D:Rifr-1). Development occurred in the absence of antibiotics, and the progeny were subjected to selection for OfxrRifrrecombinants. The parental strains differed at many polymorphic nucleotide sites, and DNA sequencing was used to map genetic crossovers and to determine the parental sources of DNA segments in 14 recombinants. Depending on the assumed DNA donor, the estimated minimal length of the transferred DNA was ≥123 kb in one recombinant but was ≥336 to ≥790 kb in all other recombinants. Such trans-DNA lengths have been associated only with conjugation in known microbial LGT systems, but natural DNA transformation remains a conceivable mechanism. LGT studies can now be performed with diverse combinations ofC. trachomatisstrains, and they could have evolutionary interest and yield useful recombinants for functional analysis of allelic differences between strains.
Affinity Isolation and I-DIRT Mass Spectrometric Analysis of the
Escherichia coli
O157:H7 Sakai RNA Polymerase Complex
Authors: Lee David J. , Busby Stephen J. W. , Westblade Lars F. , Chait Brian T.
Published: December 2007

Bacteria contain a single multisubunit RNA polymerase that is responsible for the synthesis of all RNA. Previous studies of the
Escherichia coli
K-12 laboratory strain identified a group of effector proteins that interact directly with RNA polymerase to modulate the efficiency of transcription initiation, elongation, or termination. Here we used a rapid affinity isolation technique to isolate RNA polymerase from the pathogenic
Escherichia coli
strain O157:H7 Sakai. We analyzed the RNA polymerase enzyme complex using mass spectrometry and identified associated proteins. Although
E. coli
O157:H7 Sakai contains more than 1,600 genes not present in the K-12 strain, many of which are predicted to be involved in transcription regulation, all of the identified proteins in this study were encoded on the “core”
E. coli
genome.
Genetics for
Pseudoalteromonas
Provides Tools To Manipulate Marine Bacterial Virus PM2
Authors: Kivelä Hanna M. , Madonna Stefania , Krupovìč Mart , Tutino M. Luisa , Bamford Jaana K. H.
Published: December 2007

The genetic manipulation of marine double-stranded DNA (dsDNA) bacteriophage PM2 (
Corticoviridae
) has been limited so far. The isolation of an autonomously replicating DNA element of
Pseudoalteromonas haloplanktis
TAC125 and construction of a shuttle vector replicating in both
Escherichia coli
and
Pseudoalteromonas
enabled us to design a set of conjugative shuttle plasmids encoding tRNA suppressors for amber mutations. Using a host strain carrying a suppressor plasmid allows the introduction and analysis of nonsense mutations in PM2. Here, we describe the isolation and characterization of a suppressor-sensitive PM2
sus2
mutant deficient in the structural protein P10. To infect and replicate, PM2 delivers its 10-kbp genome across the cell envelopes of two gram-negative
Pseudoalteromonas
species. The events leading to the internalization of the circular supercoiled dsDNA are puzzling. In a poorly understood process that follows receptor recognition, the virion capsid disassembles and the internal membrane fuses with the host outer membrane. While beginning to unravel the mechanism of this process, we found that protein P10 plays an essential role in the host cell penetration.
Role of the Multidrug Resistance Regulator MarA in Global Regulation of the
hdeAB
Acid Resistance Operon in
Escherichia coli
Authors: Ruiz Cristian , McMurry Laura M. , Levy Stuart B.
Published: December 2007

MarA, a transcriptional regulator in
Escherichia coli
, affects functions such as multiple-antibiotic resistance (Mar) and virulence. Usually an activator, MarA is a repressor of
hdeAB
and other acid resistance genes. We found that, in wild-type cells grown in LB medium at pH 7.0 or pH 5.5, repression of
hdeAB
by MarA occurred only in stationary phase and was reduced in the absence of H-NS and GadE, the main regulators of
hdeAB
. Moreover, repression of
hdeAB
by MarA was greater in the absence of GadX or Lrp in exponential phase at pH 7.0 and in the absence of GadW or RpoS in stationary phase at pH 5.5. In turn, MarA enhanced repression of
hdeAB
by H-NS and hindered activation by GadE in stationary phase and also reduced the activity of GadX, GadW, RpoS, and Lrp on
hdeAB
under some conditions. As a result of its direct and indirect effects, overexpression of MarA prevented most of the induction of
hdeAB
expression as cells entered stationary phase and made the cells sevenfold more sensitive to acid challenge at pH 2.5. These findings show that repression of
hdeAB
by MarA depends on pH, growth phase, and other regulators of
hdeAB
and is associated with reduced resistance to acid conditions.
Clostridium perfringens
Spore Germination: Characterization of Germinants and Their Receptors
Authors: Paredes-Sabja Daniel , Torres J. Antonio , Setlow Peter , Sarker Mahfuzur R.
Published: December 2007

Clostridium perfringens
food poisoning is caused by type A isolates carrying a chromosomal enterotoxin (
cpe
) gene (C-
cpe
), while
C. perfringens
-associated non-food-borne gastrointestinal (GI) diseases are caused by isolates carrying a plasmid-borne
cpe
gene (P-
cpe
).
C. perfringens
spores are thought to be the important infectious cell morphotype, and after inoculation into a suitable host, these spores must germinate and return to active growth to cause GI disease. We have found differences in the germination of spores of C-
cpe
and P-
cpe
isolates in that (i) while a mixture of
l
-asparagine and KCl was a good germinant for spores of C-
cpe
and P-
cpe
isolates, KCl and, to a lesser extent,
l
-asparagine triggered spore germination in C-
cpe
isolates only; and (ii)
l
-alanine or
l
-valine induced significant germination of spores of P-
cpe
but not C-
cpe
isolates. Spores of a
gerK
mutant of a C-
cpe
isolate in which two of the proteins of a spore nutrient germinant receptor were absent germinated slower than wild-type spores with KCl, did not germinate with
l
-asparagine, and germinated poorly compared to wild-type spores with the nonnutrient germinants dodecylamine and a 1:1 chelate of Ca
2+
and dipicolinic acid. In contrast, spores of a
gerAA
mutant of a C-
cpe
isolate that lacked another component of a nutrient germinant receptor germinated at the same rate as that of wild-type spores with high concentrations of KCl, although they germinated slightly slower with a lower KCl concentration, suggesting an auxiliary role for GerAA in
C. perfringens
spore germination. In sum, this study identified nutrient germinants for spores of both C-
cpe
and P-
cpe
isolates of
C. perfringens
and provided evidence that proteins encoded by the
gerK
operon are required for both nutrient-induced and non-nutrient-induced spore germination.
Differential Regulation of High-Affinity Phosphate Transport Systems of
Mycobacterium smegmatis
: Identification of PhnF, a Repressor of the
phnDCE
Operon
Authors: Gebhard Susanne , Cook Gregory M.
Published: December 2007

The uptake of phosphate into the cell via high-affinity, phosphate-specific transport systems has been studied with several species of mycobacteria. All of these species have been shown to contain several copies of such transport systems, which are synthesized in response to phosphate limitation. However, the mechanisms leading to the expression of the genes encoding these transporters have not been studied. This study reports on the investigation of the regulation of the
pstSCAB
and the
phnDCE
operons of
Mycobacterium smegmatis
. The
phn
locus contains an additional gene,
phnF
, encoding a GntR-like transcriptional regulator. Expression analyses of a
phnF
deletion mutant demonstrated that PhnF acts as a repressor of the
phnDCE
operon but does not affect the expression of
pstSCAB
. The deletion of
pstS
, which is thought to cause the constitutive expression of genes regulated by the two-component system SenX3-RegX3, led to the constitutive expression of the transcriptional fusions
pstS
-
lacZ, phnD
-
lacZ
, and
phnF
-
lacZ
, suggesting that
phnDCE
and
phnF
are conceivably new members of the SenX3-RegX3 regulon of
M. smegmatis
. Two presumptive binding sites for PhnF in the intergenic region between
phnD
and
phnF
were identified and shown to be required for the repression of
phnD
and
phnF
, respectively. We propose a model in which the transcription of
pstSCAB
is controlled by the two-component SenX3-RegX3 system, while
phnDCE
and
phnF
are subject to dual control by SenX3-RegX3 and PhnF.
Genetic and Biochemical Analysis of CodY-Binding Sites inBacillus subtilis
Authors: Belitsky Boris R. , Sonenshein Abraham L.
Published: December 2007

CodY is a global transcriptional regulator that is known to control directly the expression of at least two dozen operons inBacillus subtilis, but the rules that govern the binding of CodY to its target DNA have been unclear. Using DNase I footprinting experiments, we identified CodY-binding sites upstream of theB. subtilis ylmAandyurPgenes. The protected regions overlapped versions of a previously proposed CodY-binding consensus motif, AATTTTCWGAAAATT. Multiple single mutations were introduced into the CodY-binding sites of theylmA, yurP, dppA, andilvBgenes. The mutations affected both the affinity of CodY for its binding sites in vitro and the expression in vivo oflacZfusions that carry these mutations in their promoter regions. Our results show that versions of the AATTTTCWGAAAATT motif, first identified forLactococcus lactisCodY, with up to five mismatches play an important role in the interaction ofB. subtilisCodY with DNA.
A Combination ofsbmAandtolCMutations inEscherichia coliK-12 Tn10-Carrying Strains Results in Hypersusceptibility to Tetracycline
Authors: de Cristóbal Ricardo E. , Vincent Paula A. , Salomón Raúl A.
Published: December 2007

Previously, we demonstrated thatEscherichia coli tolCmutations reduce the high-level resistance to tetracycline afforded by the transposon Tn10-encoded TetA pump from resistance at 200 μg/ml to resistance at 40 μg/ml. In this study, we found that the addition of ansbmAmutation to atolC::Tn10mutant exacerbates this phenotype: the double mutant did not form colonies, even in the presence of tetracycline at a concentration as low as 5 μg/ml. Inactivation ofsbmAalone partially inhibited high-level tetracycline resistance, from resistance at 200 μg/ml to resistance at 120 μg/ml. There thus appears to be an additive effect of the mutations, resulting in almost complete suppression of the phenotypic expression of Tn10tetracycline resistance.
Preparation of Androstanes Related to Aphidicolin
Authors: Uyanik Cavit , Yildirim Kudret , Malay Aslihan , Hanson James R. , Hitchcock Peter B.
Published: December 2007

The preparation of 3α,4α,17β-trihydroxy-, 3α,4α,16α,17α- and 2α,3α,16α,17α-tetrahydroxy-5α-androstane derivatives (5, 11, 18) from testosterone as steroidal analogues of diterpenoid inhibitor of DNA polymerase α, aphidicolin, is described. The crystal structure of 5α-androstane-3α,4α,17β-triyl triacetate (6) is reported.
Factors affecting infection of sunflowers by Plasmopara halstedii
Authors: Cohen Yigal , Sackston W. E.
Published: December 2007

Systemic infection of sunflowers by Plasmopara halstedii occurs much more readily through hypocotyls than through roots, and through apical buds than through leaves.Systemic symptoms rarely develop from belowground infections of plants past the four-leaf stage, and from apical bud infections of plants past the eight-leaf stage. Necrotic lesions may occur and the pathogen may sporulate on hypocotyls and roots of plants with no typical systemic symptoms.Infection by sporangia produces local lesions in young leaves; some of these infections may become systemic.Immersion of 3-day-old seedlings in inoculum for 1 min resulted in some infection; highest infection resulted from 3- to 6-h exposure.Systemic infection resulted from apical bud inoculation with suspensions averaging one sporangium per plant.Optimum temperature during inoculation of 3-day-old seedlings was 15°, and during incubation was 20° to 25 °C. Optimum temperature during bud inoculation was 15°; incubation temperatures from 15° to 25 °C gave essentially similar results.
Effect on soil arthropods 1 year after application of chloropicrin to control laminated root rot. III Treatment effects on nontarget soil invertebrates
Authors: Moldenke Andrew R. , Thies Walter G.
Published: December 2007

Soil arthropods were observed as nontarget bioindicators of chloropicrin fumigation used to control Phellinus in Douglas-fir (Pseudotsugamenziesii (Mirb.) Franco) forests of Washington. Three dosages were investigated: 100% and 20% labeled dosages and a 0% control. Area-wide random samples from treated regions revealed few statistically significant (ANOVA, p < 0.05) shifts in abundance of functional guilds (one of eight). Treatment responses were shown by 23 species, a similar number decreased or increased along with increasing dosage. Stump-centered samples (0.5–2.0 m from trunk; "worst case scenarios") revealed significant changes (p < 0.05) in six of eight functional guilds of soil arthropods; most guilds were more abundant in the control and least abundant under 20% labeled dosage. Many common fungivorous oribatid mite species (20 of 63) showed significant stump-centered responses but most springtails (Collembola; 13 of 15) did not. Ordination analyses of random samples showed no area-wide effect of treatment dosage, whereas stump-centered samples revealed that the species compositions of treated versus nontreated communities of predaceous mites, oribatid mites, and endeostigmatid mites were distinct.
Adjusting volume table estimates using normal form quotient
Authors: Turnblom Eric C. , Burk Thomas E.
Published: December 2007

Total cubic volume is often of interest in estimating tree growth on remeasured permanent sample plots. A simple method to adjust the Lake States composite volume table for individual observed tree form quotients (local conditions) is presented. An adjusted form factor is found by appropriate conditioning of coefficients in a volume equation–taper equation system. Although the method is applied using the formula for the Lake States composite volume table, the methodology is quite general and may be used with other volume equation–taper equation systems whenever local adjustments to tree form are needed.
Influence of thawing rate and fungal infection by Rhizosphaera kalkhoffii on freezing injury in red spruce (Picea rubens) needles
Authors: Manter Daniel K. , Livingston William H.
Published: December 2007

Red spruce (Picea rubens Sarg.) decline has been observed in northeastern North America for the last 30 years. A major inciting stress involved in this decline is freezing injury of foliage. The objectives of this study were the following: (i) to examine how photosynthesis, needle electrolyte leakage, chlorophyll loss, needle reddening, needle loss and bud break respond to single freezing events down to −45 °C on 3-year-old seedlings; (ii) to test if faster thawing rates increase the amount of freezing injury; and (iii) to measure how Rhizosphaera kalkhoffii Bubák inoculations interact with freeze-injured needles. Two trials, one of 60 seedlings and one of 80 seedlings, were conducted. The second trial had half the seedlings covered with plastic bags for doubling the thawing time. Photosynthesis, as measured by gas exchange, was consistently the most sensitive measure, detecting nonvisible injury on uncovered seedlings (p < 0.05) at −25 °C. Measurements detecting freezing damage on covered, slower thawing seedlings were photosynthesis, chlorophyll loss, and percent budbreak. Faster thawing rates increased the amount of injury ca. 2- to 3-fold after freezing to −35 or −45 °C for all measures. Infection by R. kalkhoffii increased 40–83% after freezing needles to −40 or −45 °C. Fungal inoculations caused ca. 40–60% reduction in photosynthesis on needles frozen to −40 or −45 °C. This study suggests that two new factors can increase freezing injury on red spruce needles: a faster thawing rate and fungal (R. kalkhoffii) infection. These results are consistent with the growing knowledge that freezing injury is a complex phenomenon in red spruce.
Seasonal patterns of light transmission through boreal mixedwood canopies
Authors: Constabel A.J. , Lieffers V.J.
Published: December 2007

Measurements of light transmission were made at three heights (ground, 0.5 m, and 1.3 m) in six stands in each of three overstory types: pure young aspen (Populustremuloides Michx.); pure old aspen; and mixed old aspen–white spruce (Piceaglauca (Moench) Voss). Light transmission was measured in the spring, summer, and autumn. In the summer, the amount of light that reached the forest floor was very low in all overstory types (5.9% of incident light). In the mixed stands, the overstory intercepted 87% of above-canopy light, and the understory intercepted 52% of its above-canopy light. In the old aspen stands, there was more light transmission through the overstory, which led to increased understory cover and light interception at that level. In the autumn, 26% of light reached the forest floor in the pure aspen stands because of the loss of deciduous over- and understory foliage. In the mixed stands only 8% of above-overstory light reached the forest floor in the autumn. The deciduous leaf-off period in the spring with relatively high solar elevation angles showed the greatest light transmission through the overstory, ranging from 22% in the old mixedwood to 59% in the young aspen stand. Findings suggest that white spruce seedlings less than 0.5 m in height may be at or below the photosynthetic light compensation point for much of the summer. High light during the deciduous leaf-off periods in the spring and autumn may account for a large portion of their annual carbon gain.
Inbreeding effects on the spring frost hardiness of coastal Douglas-fir
Authors: Shortt R.L. , Hawkins B.J. , Woods J.H.
Published: December 2007

An experiment was designed to investigate the effect of inbreeding on frost hardiness of coastal Douglas-fir (Pseudotsugamenziesii (Mirb.) Franco var. menziesii). Frost hardiness of branch samples from 19 families of Douglas-fir (four inbreeding levels within five common ancestor groups) was assessed using freeze-induced electrolyte leakage and visual assessment methods in February and April 1993. Frost hardiness of clones of the common ancestors was assessed again in February 1994 using the freeze-induced electrolyte leakage method. There was a significant correlation between the results of the two methods of frost hardiness assessment. Neither method showed a consistent relationship between frost hardiness and inbreeding level, and within-family variability in hardiness was high. Significant differences in hardiness among the common ancestor groups were evident.
Site regions revisited: a climatic analysis of Hills' site regions for the province of Ontario using a parametric method
Authors: Mackey Brendan G. , McKenney Daniel W. , Yang Yin-Qian , McMahon June P. , Hutchinson Michael F.
Published: December 2007

not available
Corky root disease of Douglas-fir seedlings: pathogenicity of the nematode Xiphinemabakeri alone and in combination with the fungus Cylindrocarpondestructans
Authors: Sutherland Jack R.
Published: December 2007

Growth-room studies were made to determine both, alone and combined, the pathogenicity of the nematode Xiphinemabakeri Williams and the fungus Cylindrocarpondestructans (Zinnsm.) Scholten to Douglas-fir [Pseudotsugamenziesii (Mirb.) Franco] seedlings. Inoculations with 20, 40, and 60 X. bakeri nematodes per seedling resulted in nematode population increases and development of typical corky root disease symptoms. Cylindrocarpondestructans was not pathogenic to seedlings; no consistent synergistic effects were detected when inoculations were made with the nematode and fungus together. It was concluded that X. bakeri is the 'primary' pathogen in corky root etiology.
Motion-induced growth effects in Douglas fir
Authors: Kellogg R. M. , Steucek G. L.
Published: December 2007

Daily brief intervals (30 s) of relatively mild shaking had a marked influence on leader growth of young Douglas-fir (Pseudotsugamenziesii (Mirb.) Franco) stems grown in a greenhouse. When the treatment was applied to naturally wind-loaded stems, there was some suggestion that height growth was reduced more than the considerable reduction from wind action alone. Neither the pattern nor magnitude of xylem growth appeared to be affected by the treatment, whereas wind action produced a significant change in the pattern. It is suggested that the response to wind and shaking stimuli may differ.
Photosynthate allocation in Pinustaeda. I. Substrate requirements for synthesis of shoot biomass
Authors: Chung Hsu-Ho , Barnes Robert L.
Published: December 2007

Procedures were developed to estimate the amount of glucose substrate required to biosynthesize shoot biomass of Pinustaeda L. By using compositional data from our own analyses and from the literature, glucose requirements per gram of major biomass constituent fractions were calculated to be as follows: lipids, 3.019 g; phenolics, 1.919 g; lignin, 1.897 g; nitrogenous compounds, 1.579 g; organic acids, 1.484 g; and carbohydrates, 1.182 g. From determinations of shoot weight and composition we calculated that 1.574 g of glucose was required to produce 1 g of typical shoot biomass.The computational procedures were also applied to analytical data from four other tree species, with glucose requirements estimated to range from 1.216 g for the cambial zone of Eucalyptusregnans F. Muell. to 1.603 g for bark of Pinuselliottii Englm.Application of our findings to studies of biomass formation of forest trees are discussed.
Water relations and drought resistance of young Pinusbanksiana and P. resinosa plantation trees
Authors: Pereira J. S. , Kozlowski T. T.
Published: December 2007

Daytime changes in needle water potential and needle diffusion resistance of young Pinusbanksiana and P. resinosa plantation trees were determined at various times throughout the 1974 growing season in northern Wisconsin. Early in the growing season, early-morning water potential and daytime needle diffusion resistance varied little between the two species. However, midday water potentials generally were higher in P. banksiana than in P. resinosa. As the season progressed, water potentials in the early morning were lower, and they decreased more during the day in P. resinosa than in P. banksiana. In addition, stomata of P. banksiana usually closed earlier in the day. Pinusbanksiana appeared to avoid drought better than P. resinosa because the former maintained a higher needle water potential throughout most of the growing season. This reflected lower transpiration and slower soil water depletion associated with lower leaf area and stomatal closure at higher needle water potential in P. banksiana.
Influence of cutting methods on natural and artificial regeneration of yellow birch in Quebec northern hardwoods
Authors: Roberge M. R.
Published: December 2007

This is an interim assessment of a thinning and of a group-selection cutting associated with various treatments to increase yellow birch (Betulaalleghaniensis Britton) regeneration and production. Ten years after cutting, improved quality, health, and growth rate of trees were observed where thinning had been carried out with a very abundant regeneration which was growing well in clear-cut patches. Ground scarification with or without artificial seeding stimulated yellow birch establishment, but the effects of stimulation did not last and had disappeared 5 years after scarification. Only planting resulted in a notable increase in stocking of dominant yellow birch still conspicuous 5 and 10 years after clear-cutting and in a percentage of yellow birch stems in the main story superior to that of the original stand.
A comparison of rooting success of greenhouse-grown and field-grown slash pine cuttings
Authors: Bower R. , Buijtenen J. P. van
Published: December 2007

This experiment compared the rooting response of cuttings taken monthly from slash pine (Pinuselliottii Engelm.) clones maintained in a greenhouse environment and in a field environment. Greenhouse cuttings rooted better in all months except for the first May collection. Seasonal variation was less pronounced in cuttings taken from the greenhouse-maintained plants, and May collections gave the highest percentage rooted from both locations. Average rooting by clone varied from 47 to 4% for field tests compared with 63 to 19% for greenhouse tests. Environmental effects on rooting, known to be critical on ramets in the propagating bed, are shown to be important also on the ortets.
Particulate matter air pollution exposure promotes recruitment of monocytes into atherosclerotic plaques
Authors: Yatera Kazuhiro , Hsieh Joanne , Hogg James C. , Tranfield Erin , Suzuki Hisashi , Shih Chih-Horng , Behzad Ali R. , Vincent Renaud , van Eeden Stephan F.
Published: December 2007

Epidemiologic studies have shown an association between exposure to ambient particulate air pollution <10 μm in diameter (PM10) and increased cardiovascular morbidity and mortality. We previously showed that PM10 exposure causes progression of atherosclerosis in coronary arteries. We postulate that the recruitment of monocytes from the circulation into atherosclerotic lesions is a key step in this PM10-induced acceleration of atherosclerosis. The study objective was to quantify the recruitment of circulating monocytes into vessel walls and the progression of atherosclerotic plaques induced by exposure to PM10. Female Watanabe heritable hyperlipidemic rabbits, which naturally develop systemic atherosclerosis, were exposed to PM10 (EHC-93) or vehicle by intratracheal instillation twice a week for 4 wk. Monocytes, labeled with 5-bromo-2′-deoxyuridine (BrdU) in donors, were transfused to recipient rabbits as whole blood, and the recruitment of BrdU-labeled cells into vessel walls and plaques in recipients was measured by quantitative histological methodology. Exposure to PM10 caused progression of atherosclerotic lesions in thoracic and abdominal aorta. It also decreased circulating monocyte counts, decreased circulating monocytes expressing high levels of CD31 (platelet endothelial cell adhesion molecule-1) and CD49d (very late antigen-4 α-chain), and increased expression of CD54 (ICAM-1) and CD106 (VCAM-1) in plaques. Exposure to PM10 increased the number of BrdU-labeled monocytes adherent to endothelium over plaques and increased the migration of BrdU-labeled monocytes into plaques and smooth muscle underneath plaques. We conclude that exposure to ambient air pollution particles promotes the recruitment of circulating monocytes into atherosclerotic plaques and speculate that this is a critically important step in the PM10-induced progression of atherosclerosis.
Prevention of HIF-1 activation and iNOS gene targeting by low-dose cadmium results in loss of myocardial hypoxic preconditioning in the rat
Authors: Belaidi Elise , Beguin Pauline C. , Levy Patrick , Ribuot Christophe , Godin-Ribuot Diane
Published: December 2007

This study aimed to underline the interaction between hypoxia-inducible factor-1 (HIF-1) and the inducible nitric oxide synthase (iNOS) gene in vivo and their contribution to the delayed myocardial preconditioning induced by acute intermittent hypoxia (IH) in the rat using chromatin immunoprecipitation and pharmacological inhibition by low-dose cadmium. Langendorff-perfused hearts of Wistar rats exposed to normoxia or IH 24 h earlier were submitted to global ischemia and reperfusion. Effects of iNOS inhibition by aminoguanidine (100 μM) before ischemia or of low-dose injection of cadmium chloride (1 mg/kg) before normoxia or IH were tested. Myocardial HIF-1 and iNOS quantification and in vivo chromatin immunoprecipitation of HIF-1 bound to the iNOS gene promoter were performed. IH-induced delayed cardioprotection resulted in an improvement in coronary flow and functional recovery at reperfusion and a decrease in infarct size. Myocardial HIF-1 activity was increased with resulting targeting of the iNOS gene. Aminoguanidine abolished the cardioprotective effects of IH. Cadmium chloride treatment before IH prevented myocardial HIF-1 activation (72.3 ± 4.0 vs. 42.1 ± 9.7 arbitrary units after cadmium chloride; P < 0.05), targeting of the iNOS gene, iNOS expression, and preconditioning (infarct size: 15.9 ± 5.6 vs. 30.1 ± 5.4% after cadmium chloride; P < 0.05). This study is the first to demonstrate the interaction of HIF-1 with the myocardial iNOS gene in situ after hypoxic preconditioning. Prevention of HIF-1 activation and iNOS gene targeting by a single low dose of cadmium abolished the delayed cardioprotective effects, bringing insight into the cardiovascular consequences of cadmium exposure.
Overexpression of pyruvate dehydrogenase kinase 4 in heart perturbs metabolism and exacerbates calcineurin-induced cardiomyopathy
Authors: Zhao Guixiang , Jeoung Nam Ho , Burgess Shawn C. , Rosaaen-Stowe Kimberly A. , Inagaki Takeshi , Latif Shuaib , Shelton John M. , McAnally John , Bassel-Duby Rhonda , Harris Robert A. , Richardson James A. , Kliewer Steven A.
Published: December 2007

The heart adapts to changes in nutritional status and energy demands by adjusting its relative metabolism of carbohydrates and fatty acids. Loss of this metabolic flexibility such as occurs in diabetes mellitus is associated with cardiovascular disease and heart failure. To study the long-term consequences of impaired metabolic flexibility, we have generated mice that overexpress pyruvate dehydrogenase kinase (PDK)4 selectively in the heart. Hearts from PDK4 transgenic mice have a marked decrease in glucose oxidation and a corresponding increase in fatty acid catabolism. Although no overt cardiomyopathy was observed in the PDK4 transgenic mice, introduction of the PDK4 transgene into mice expressing a constitutively active form of the phosphatase calcineurin, which causes cardiac hypertrophy, caused cardiomyocyte fibrosis and a striking increase in mortality. These results demonstrate that cardiac-specific overexpression of PDK4 is sufficient to cause a loss of metabolic flexibility that exacerbates cardiomyopathy caused by the calcineurin stress-activated pathway.
Regulation of contractility by Hsp27 and Hic-5 in rat mesenteric small arteries
Authors: Srinivasan R. , Forman S. , Quinlan R. A. , Ohanian J. , Ohanian V.
Published: December 2007

The regulation of small artery contractility by vasoconstrictors is important for vascular function, and actin cytoskeleton remodeling is required for contraction. p38 MAPK and tyrosine kinases are implicated in actin polymerization and contraction through heat shock protein 27 (Hsp27) and the cytoskeletal protein paxillin, respectively. We evaluated the roles of downstream targets of p38 MAPK and tyrosine kinases in cytoskeletal reorganization and contraction and whether the two signaling pathways regulate contraction independent of each other. We identified the expression of the paxillin homologue hydrogen peroxide-inducible clone-5 (Hic-5) and showed its activation by norepinephrine (NE) in a Src-dependent manner. Furthermore, we demonstrated a NE-induced interaction of proline-rich tyrosine kinase-2 (PYK2) but not Src or p125 focal adhesion kinase with Hic-5. This interaction was Src dependent, suggesting that Hic-5 was a substrate for PYK2 downstream from Src. The activation of Hic-5 induced its relocalization to the cytosol. The parallel activation of Hsp27 by NE was p38 MAPK dependent and led to its dissociation from actin filaments and translocation from membrane to cytosol and increased actin polymerization. Both Hsp27 and Hic-5 activation resulted in their association within the same time frame as NE-induced contraction, and the inhibition of either p38 MAPK or Src inhibited the interaction between Hsp27 and Hic-5 and the contractile response. Furthermore, combined p38 MAPK and Src inhibition had no greater effect on contraction than individual inhibition, suggesting that the two pathways act through a common mechanism. These data show that NE-induced activation and the association of Hsp27 and Hic-5 are required for the reorganization of the actin cytoskeleton and force development in small arteries.
Dysregulation of dopamine-dependent mechanisms as a determinant of hypertension: studies in dopamine receptor knockout mice
Authors: Zeng Chunyu , Armando Ines , Luo Yingjin , Eisner Gilbert M. , Felder Robin A. , Jose Pedro A.
Published: December 2007

Dopamine plays an important role in the pathogenesis of hypertension by regulating epithelial sodium transport and by interacting with vasoactive hormones/humoral factors, such as aldosterone, angiotensin, catecholamines, endothelin, oxytocin, prolactin pro-opiomelancortin, reactive oxygen species, renin, and vasopressin. Dopamine receptors are classified into D1-like (D1and D5) and D2-like (D2, D3, and D4) subtypes based on their structure and pharmacology. In recent years, mice deficient in one or more of the five dopamine receptor subtypes have been generated, leading to a better understanding of the physiological role of each of the dopamine receptor subtypes. This review summarizes the results from studies of various dopamine receptor mutant mice on the role of individual dopamine receptor subtypes and their interactions with other G protein-coupled receptors in the regulation of blood pressure.
Cardioprotection with palm oil tocotrienols: comparision of different isomers
Authors: Das Samarjit , Lekli Istvan , Das Manika , Szabo Gergo , Varadi Judit , Juhasz Bela , Bak Istvan , Nesaretam Kalanithi , Tosaki Arpad , Powell Saul R. , Das Dipak K.
Published: December 2007

A recent study from our laboratory indicated the cardioprotective ability of the tocotrienol-rich fraction (TRF) from red palm oil. The present study compared cardioprotective abilities of different isomers of tocotrienol against TRF as recently tocotrienol has been found to function as a potent neuroprotective agent against stroke. Rats were randomly assigned to one of the following groups: animals were given, by gavage, either 0.35%, 1%, or 3.5% TRF for two different periods of time (2 or 4 wk) or 0.03, 0.3, and 3 mg/kg body wt of one of the isomers of tocotrienol (α, γ, or δ) for 4 wk; control animals were given, by gavage, vehicle only. After 2 or 4 wk, rats were killed, and their hearts were then subjected to 30 min of global ischemia followed by 2 h of reperfusion. Dose-response and time-response experiments revealed that the optimal concentration for TRF was 3.5% TRF and 0.3 mg/kg body wt of tocotrienol given for 4 wk. TRF as well as all the isomers of tocotrienol used in our study provided cardioprotection, as evidenced by their ability to improve postischemic ventricular function and reduce myocardial infarct size. The γ-isoform of tocotrienol was the most cardioprotective of all the isomers followed by the α- and δ-isoforms. The molecular mechanisms of cardioprotection afforded by tocotrienol isoforms were probed by evaluating their respective abilities to stabilize the proteasome, allowing it to maintain a balance between prodeath and prosurvival signals. Our results demonstrated that tocotrienol isoforms reduced c-Src but increased the phosphorylation of Akt, thus generating a survival signal.
Exercise induces a cardiac mitochondrial phenotype that resists apoptotic stimuli
Authors: Kavazis Andreas N. , McClung Joseph M. , Hood David A. , Powers Scott K.
Published: December 2007

Ischemia-reperfusion-induced calcium overload and production of reactive oxygen species can trigger apoptosis by promoting the release of proapoptotic factors via the mitochondrial permeability transition pore. While it is clear that endurance exercise provides cardioprotection against ischemia-reperfusion-induced injury, it is unknown if exercise training directly alters mitochondria phenotype and confers protection against apoptotic stimuli in both subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. We hypothesized that exercise training increases expression of endogenous antioxidant enzymes and other antiapoptotic proteins, resulting in a SS and IMF mitochondrial phenotype that resists apoptotic stimuli. Mitochondria isolated from hearts of sedentary ( n = 8) and exercised-trained ( n = 8) adult male rats were studied. Endurance exercise increased the protein levels of primary antioxidant enzymes in both SS and IMF mitochondria. Furthermore, exercise increased the levels of antiapoptotic proteins in the heart, including the apoptosis repressor with a caspase recruitment domain and inducible heat shock protein 70. Importantly, our findings reveal that endurance exercise training attenuates reactive oxygen species-induced cytochrome c release from heart mitochondria. These changes are accompanied by a lower maximal rate of mitochondrial permeability transition pore opening ( Vmax) and prolonged time to Vmaxin both SS and IMF cardiac mitochondria. These novel findings reveal that endurance exercise promotes biochemical alterations in cardiac SS and IMF mitochondria, resulting in a phenotype that resists apoptotic stimuli. Furthermore, these results are consistent with the concept that exercise-induced mitochondrial adaptations contribute to exercise-induced cardioprotection.
Integrated multimodal-catheter imaging unveils principal relationships among ventricular electrical activity, anatomy, and function
Authors: Rao Liyun , Ling Yuesheng , He Renjie , Gilbert April L. , Frangogiannis Nikolaos G. , Wang Jianwen , Nagueh Sherif F. , Khoury Dirar S.
Published: December 2007

Multiple imaging modalities are employed independent of one another while managing complex cardiac arrhythmias. To combine electrical, anatomical, and functional imaging in a single catheter system, we developed a balloon catheter that carried 64 electrodes on its surface and an intracardiac echocardiography (ICE) catheter through a central lumen. The catheter system was inserted, and the balloon was inflated inside the left ventricle (LV) of eight dogs with 6-wk-old infarction, created by occlusion in the left anterior descending coronary artery. Anatomy was constructed by ICE imaging (9 MHz) through the balloon. Single-beat noncontact mapping (NCM) was performed via the multielectrode array to reconstruct unipolar endocardial electrograms during sinus rhythm. Standard contact mapping (CM) of the endocardium was also carried out for reference. Myocardial infarction in anterior LV extending from the middle to apical regions was localized both by ICE and NCM and validated by CM and pathology. The overall difference in the activation times between NCM and CM was 3 ± 1 ms. Unipolar voltage in infarcted middle anterior LV was smaller than the voltage in normal middle inferior LV both by NCM (11 ± 4 vs. 16 ± 3 mV; P = 0.002) and CM (11 ± 3 vs. 20 ± 4 mV; P < 0.001). Unipolar voltage was also inversely related to infarct transmurality, both by NCM ( r = −0.87; P = 0.005) and CM ( r = −0.94; P < 0.001). The infarct area by ICE (7.7 ± 2.9 cm2) was in agreement with CM (bipolar voltage, <1 mV; and area, 7.6 ± 3.3 cm2; r = 0.80; P = 0.016). Meanwhile, the voltage threshold that depicted the infarct area by NCM was directly related to the smallest unipolar voltage reconstructed within the infarct ( r = 0.96; P < 0.001). In conclusion, combining NCM and ICE imaging in a single catheter system is feasible. The preclinical development of such an integrated system and its evaluation in experimental myocardial infarction demonstrate capabilities for single-beat mapping at multiple sites as well as the online assessment of anatomy and myocardial function.
Mitochondrial metabolism, redox signaling, and fusion: a mitochondria-ROS-HIF-1α-Kv1.5 O2-sensing pathway at the intersection of pulmonary hypertension and cancer
Authors: Archer Stephen L. , Gomberg-Maitland Mardi , Maitland Michael L. , Rich Stuart , Garcia Joe G. N. , Weir E. Kenneth
Published: December 2007

Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by vascular obstruction and right ventricular failure. Although the fundamental cause remains elusive, many predisposing and disease-modifying abnormalities occur, including endothelial injury/dysfunction, bone morphogenetic protein receptor-2 gene mutations, decreased expression of the O2-sensitive K+channel (Kv1.5), transcription factor activation [hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-activating T cells], de novo expression of survivin, and increased expression/activity of both serotonin transporters and platelet-derived growth factor receptors. Together, these abnormalities create a cancerlike, proliferative, apoptosis-resistant phenotype in pulmonary artery smooth muscle cells (PASMCs). A possible unifying mechanism for PAH comes from studies of fawn-hooded rats, which manifest spontaneous PAH and impaired O2sensing. PASMC mitochondria normally produce reactive O2species (ROS) in proportion to Po2. Superoxide dismutase 2 (SOD2) converts intramitochondrial superoxide to diffusible H2O2, which serves as a redox-signaling molecule, regulating pulmonary vascular tone and structure through effects on Kv1.5 and transcription factors. O2sensing is mediated by this mitochondria-ROS-HIF-1α-Kv1.5 pathway. In PAH and cancer, mitochondrial metabolism and redox signaling are reversibly disordered, creating a pseudohypoxic redox state characterized by normoxic decreases in ROS, a shift from oxidative to glycolytic metabolism and HIF-1α activation. Three newly recognized mitochondrial abnormalities disrupt the mitochondria-ROS-HIF-1α-Kv1.5 pathway: 1) mitochondrial pyruvate dehydrogenase kinase activation, 2) SOD2 deficiency, and 3) fragmentation and/or hyperpolarization of the mitochondrial reticulum. The pyruvate dehydrogenase kinase inhibitor, dichloroacetate, corrects the mitochondrial abnormalities in experimental models of PAH and human cancer, causing a regression of both diseases. Mitochondrial abnormalities that disturb the ROS-HIF-1α-Kv1.5 O2-sensing pathway contribute to the pathogenesis of PAH and cancer and constitute promising therapeutic targets.
Thrombin enhances the barrier function of rat microvascular endothelium in a PAR-1-dependent manner
Authors: Troyanovsky B. , Alvarez D. F. , King J. A. , Schaphorst K. L.
Published: December 2007

Thrombin is a multifunctional coagulation protease with pro- and anti-inflammatory vascular effects. We questioned whether thrombin may have segmentally differentiated effects on pulmonary endothelium. In cultured rat endothelial cells, rat thrombin (10 U/ml) recapitulated the previously reported decrease in transmonolayer electrical resistance (TER), F-actin stress fiber formation, paracellular gap formation, and increased permeability. In contrast, in rat pulmonary microvascular endothelial cells (PMVEC), isolated on the basis of Griffonia simplicifolia lectin recognition, thrombin increased TER, induced fewer stress fibers, and decreased permeability. To assess for differential proteinase-activated receptor (PAR) expression as a basis for the different responses, PAR family expression was analyzed. Both pulmonary artery endothelial cells and PMVEC expressed PAR-1 and PAR-2; however, only PMVEC expressed PAR-3, as shown by both RT-PCR and Western analysis. PAR-1 activating peptides (PAR-APs: SFLLRN-NH2and TFLLRN-NH2) were used to confirm a role for the PAR-1 receptor. PAR-APs (25–250 μM) also increased TER, formed fewer stress fibers, and did not induce paracellular gaps in PMVEC in contrast to that shown in pulmonary artery endothelial cells. These results were confirmed in isolated perfused rat lung preparations. PAR-APs (100 μg/ml) induced a 60% increase in the filtration coefficient over baseline. However, by transmission electron microscopy, perivascular fluid cuffs were seen only along conduit veins and arteries without evidence of intra-alveolar edema. We conclude that thrombin exerts a segmentally differentiated effect on endothelial barrier function in vitro, which corresponds to a pattern of predominant perivascular fluid cuff formation in situ. This may indicate a distinct role for thrombin in the microcirculation.
Dietary flaxseed enhances antioxidant defenses and is protective in a mouse model of lung ischemia-reperfusion injury
Authors: Lee James C. , Bhora Faiz , Sun Jing , Cheng Guanjun , Arguiri Evguenia , Solomides Charalambos C. , Chatterjee Shampa , Christofidou-Solomidou Melpo
Published: December 2007

Dietary flaxseed (FS) is a nutritional whole grain with high contents of omega-3 fatty acids and lignans with anti-inflammatory and antioxidant properties. We evaluated FS in a murine model of pulmonary ischemia-reperfusion injury (IRI) by dietary supplementation of 0% (control) or 10% (treatment) FS before IRI. Mice fed 0% FS undergoing IRI had a significant decrease in arterial oxygenation (PaO2) and a significant increase in bronchoalveolar lavage (BAL) protein compared with sham-operated mice. However, mice fed 10% FS undergoing IRI had a significant improvement in both PaO2and BAL protein compared with mice fed 0% FS undergoing IRI. In addition, oxidative lung damage was decreased in 10% FS-supplemented mice undergoing IRI, as assessed by malondialdehyde levels. Immunohistochemical staining of lungs for iPF2α-III F2isoprostane, a measure of lipid oxidation, was diminished. FS-supplemented mice had less reactive oxygen species (ROS) release from the vascular endothelium in lungs in an ex vivo model of IRI, and alveolar macrophages isolated from FS-fed mice had significantly reduced ROS generation in response to oxidative burst. Pulmonary microvascular endothelial cells produced less ROS in a flow cessation model of ischemia when preincubated with purified FS lignan metabolites. Pharmacological inhibition of heme oxygenase-1 (HO-1) resulted in only a partial reduction of FS protection in the same model. We conclude that dietary FS is protective against IRI in an experimental murine model and that FS affects ROS generation and ROS detoxification via pathways not limited to upregulation of antioxidant enzymes such as HO-1.