bluuebunny/crossref_metadata_2025 huggingface dataset:
Peptide Transport in
Helicobacter pylori
: Roles of Dpp and Opp Systems and Evidence for Additional Peptide Transporters
Authors: Weinberg Michael V. , Maier Robert J.
Published: February 2007

Despite research into the nutritional requirements of
Helicobacter pylori
, little is known regarding its use of complex substrates, such as peptides. Analysis of genome sequences revealed putative ABC-type transporter genes for dipeptide (
dppABCDF
) and oligopeptide (
oppABCD
) transport. Genes from each system were PCR amplified, cloned, and disrupted by cassette insertion either individually (
dppA, dppB, dppC, oppA, oppB
, and
oppC
) or to create double mutants (
dppA oppA, dppB oppB, dppB dppC
, and
oppB oppC
). Peptide-utilizing abilities of the strains were assessed by monitoring growth in a chemically defined medium where the only source of the essential amino acid isoleucine was from peptides of various lengths (two to nine amino acids long). The dipeptide system mutants lacked the ability to use certain dipeptides, hexapeptides, and nonapeptides. However, these mutants retained some ability to grow with other dipeptides, tripeptides, and tetrapeptides. Of the oligopeptide mutants, only the
oppB
strain differed significantly from the wild type. This strain showed a wild-type phenotype for growth with longer peptides (hexa- and nonapeptides) while having a decreased ability to utilize di-, tri-, and tetrapeptides. The
dppA oppA
and
dppB oppB
mutants showed similar phenotypes to those of the
dppA
and
dppB
mutants, respectively. Peptide digestion by metalloproteases was ruled out as the cause for residual peptide transport by growing mutant strains in the presence of either EDTA or EGTA. Degradation products associated with a fluorescein isothiocyanate-labeled hexapeptide (plus cells) were minimal. An as yet unidentified peptide transport system(s) in
H. pylori
is proposed to be responsible for the residual transport.
Quinone Reduction by the Na+-Translocating NADH Dehydrogenase Promotes Extracellular Superoxide Production inVibrio cholerae
Authors: Lin Po-Chi , Türk Karin , Häse Claudia C. , Fritz Günter , Steuber Julia
Published: February 2007

The pathogenicity ofVibrio choleraeis influenced by sodium ions which are actively extruded from the cell by the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR). To study the function of the Na+-NQR in the respiratory chain ofV. cholerae, we examined the formation of organic radicals and superoxide in a wild-type strain and a mutant strain lacking the Na+-NQR. Upon reduction with NADH, an organic radical was detected in native membranes by electron paramagnetic resonance spectroscopy which was assigned to ubisemiquinones generated by the Na+-NQR. The radical concentration increased from 0.2 mM at 0.08 mM Na+to 0.4 mM at 14.7 mM Na+, indicating that the concentration of the coupling cation influences the redox state of the quinone pool inV. choleraemembranes. During respiration,V. choleraecells produced extracellular superoxide with a specific activity of 10.2 nmol min−1mg−1in the wild type compared to 3.1 nmol min−1mg−1in the NQR deletion strain. Raising the Na+concentration from 0.1 to 5 mM increased the rate of superoxide formation in the wild-typeV. choleraestrain by at least 70%. Rates of respiratory H2O2formation by wild-typeV. choleraecells (30.9 nmol min−1mg−1) were threefold higher than rates observed with the mutant strain lacking the Na+-NQR (9.7 nmol min−1mg−1). Our study shows that environmental Na+could stimulate ubisemiquinone formation by the Na+-NQR and hereby enhance the production of reactive oxygen species formed during the autoxidation of reduced quinones.
Role of EAL-Containing Proteins in Multicellular Behavior ofSalmonella entericaSerovar Typhimurium
Authors: Simm Roger , Lusch Astrid , Kader Abdul , Andersson Mats , Römling Ute
Published: February 2007

GGDEF and EAL domain proteins are involved in turnover of the novel secondary messenger cyclic di(3′→5′)-guanylic acid (c-di-GMP) in many bacteria. The rdar morphotype, a multicellular behavior ofSalmonella entericaserovar Typhimurium characterized by the expression of the extracellular matrix components cellulose and curli fimbriae is controlled by c-di-GMP. In this work the roles of the EAL and GGDEF-EAL domain proteins on rdar morphotype development were investigated. Knockout of four of 15 EAL and GGDEF-EAL domain proteins upregulated rdar morphotype expression and expression of CsgD, the central regulator of the rdar morphotype, and partially downregulated c-di-GMP concentrations. More-detailed analysis showed that the EAL domain protein STM4264 and the GGDEF-EAL domain protein STM1703, which highly downregulated the rdar morphotype, have overlapping yet distinct functions. Another subset of EAL and GGDEF-EAL domain proteins influenced multicellular behavior in liquid culture and flagellum-mediated motility. Consequently, this work has shown that several EAL and GGDEF-EAL domain proteins, which act as phosphodiesterases, play a determinative role in the expression level of multicellular behavior ofSalmonella entericaserovar Typhimurium.
Analysis of Temporal Gene Expression duringBacillus subtilisSpore Germination and Outgrowth
Authors: Keijser Bart J. F. , Ter Beek Alex , Rauwerda Han , Schuren Frank , Montijn Roy , van der Spek Hans , Brul Stanley
Published: February 2007

Bacillus subtilisforms dormant spores upon nutrient depletion. Under favorable environmental conditions, the spore breaks its dormancy and resumes growth in a process called spore germination and outgrowth. To elucidate the physiological processes that occur during the transition of the dormant spore to an actively growing vegetative cell, we studied this process in a time-dependent manner by a combination of microscopy, analysis of extracellular metabolites, and a genome-wide analysis of transcription. The results indicate the presence of abundant levels of late sporulation transcripts in dormant spores. In addition, the results suggest the existence of a complex and well-regulated spore outgrowth program, involving the temporal expression of at least 30% of theB. subtilisgenome.
Characterization of a Novel Two-Partner Secretion System in
Escherichia coli
O157:H7
Authors: Choi Peter S. , Dawson Ashley J. , Bernstein Harris D.
Published: February 2007

Gram-negative bacteria contain multiple secretion pathways that facilitate the translocation of proteins across the outer membrane. The two-partner secretion (TPS) system is composed of two essential components, a secreted exoprotein and a pore-forming β barrel protein that is thought to transport the exoprotein across the outer membrane. A putative TPS system was previously described in the annotation of the genome of
Escherichia coli
O157:H7 strain EDL933. We found that the two components of this system, which we designate OtpA and OtpB, are not predicted to belong to either of the two major subtypes of TPS systems (hemolysins and adhesins) based on their sequences. Nevertheless, we obtained direct evidence that OtpA and OtpB constitute a bona fide TPS system. We found that secretion of OtpA into the extracellular environment in
E. coli
O157:H7 requires OtpB and that when OtpA was produced in an
E. coli
K-12 strain, its secretion was strictly dependent on the production of OtpB. Furthermore, using OtpA/OtpB as a model system, we show that protein secretion via the TPS pathway is extremely rapid.
Biofilms Formed by Nontypeable
Haemophilus influenzae
In Vivo Contain both Double-Stranded DNA and Type IV Pilin Protein
Authors: Jurcisek Joseph A. , Bakaletz Lauren O.
Published: February 2007

Nontypeable
Haemophilus influenzae
(NTHI) strains are members of the normal human nasopharyngeal flora, as well as frequent opportunistic pathogens of both the upper and lower respiratory tracts. Recently, it has been shown that NTHI can form biofilms both in vitro and in vivo. NTHI strains within in vitro-formed biofilms differentially express both epitopes of lipooligosaccharide (LOS) and the outer membrane proteins P2, P5, and P6, whereas those generated either in a 96-well plate assay in vitro or in a mammalian host have been shown to incorporate a specific glycoform of sialylated LOS within the biofilm matrix. While DNA has been identified as a key component of the biofilm matrix formed in vitro by several bacterial pathogens, here we demonstrate for the first time that in addition to sialylated LOS, the biofilm formed by NTHI in vivo contains both type IV pilin protein and a significant amount of double-stranded DNA. The DNA appeared to be arranged in a dense interlaced meshwork of fine strands as well as in individual thicker “ropes” that span water channels, suggesting that DNA could be imparting structural stability to the biofilm produced by NTHI in vivo. The presence of type IV pilin protein both appearing as small aggregates within the biofilm matrix and tracking along DNA strands supports our observations which showed that type IV pili are expressed by NTHI during experimental otitis media when these bacteria form a biofilm in the middle ear space.
Mutations Blocking Side Chain Assembly, Polymerization, or Transport of a Wzy-DependentStreptococcus pneumoniaeCapsule Are Lethal in the Absence of Suppressor Mutations and Can Affect Polymer Transfer to the Cell Wall
Authors: Xayarath Bobbi , Yother Janet
Published: February 2007

Extracellular polysaccharides of many bacteria are synthesized by the Wzy polymerase-dependent mechanism, where long-chain polymers are assembled from undecaprenyl-phosphate-linked repeat units on the outer face of the cytoplasmic membrane. In gram-positive bacteria, Wzy-dependent capsules remain largely cell associated via membrane and peptidoglycan linkages. Like many Wzy-dependent capsules, theStreptococcus pneumoniaeserotype 2 capsule is branched. In this study, we found that deletions ofcps2K,cps2J, orcps2H, which encode a UDP-glucose dehydrogenase necessary for side chain synthesis, the putative Wzx transporter (flippase), and the putative Wzy polymerase, respectively, were obtained only in the presence of suppressor mutations. Most of the suppressor mutations were incps2E, which encodes the initiating glycosyltransferase for capsule synthesis. Thecps2Kmutants containing the suppressor mutations produced low levels of high-molecular-weight polymer that was detected only in membrane fractions.cps2K-repaired mutants exhibited only modest increases in capsule production due to the effect of the secondary mutation, but capsule was detectable in both membrane and cell wall fractions. Lethality of thecps2K,cps2J, andcps2Hmutations was likely due to sequestration of undecaprenyl-phosphate in the capsule pathway and either preclusion of its turnover for utilization in essential pathways or destabilization of the membrane due to an accumulation of lipid-linked intermediates. The results demonstrate that proper polymer assembly requires not only a functional transporter and polymerase but also complete repeat units. A central role for the initiating glycosyltransferase in controlling capsule synthesis is also suggested.
Separation of Chromosome Termini during Sporulation of
Bacillus subtilis
Depends on SpoIIIE
Authors: Bogush Marina , Xenopoulos Panagiotis , Piggot Patrick J.
Published: February 2007

Bacillus subtilis
undergoes a highly distinctive division during spore formation. It yields two unequal cells, the mother cell and the prespore, and septum formation is completed before the origin-distal 70% of the chromosome has entered the smaller prespore. The mother cell subsequently engulfs the prespore. Two different probes were used to study the behavior of the terminus (
ter
) region of the chromosome during spore formation. Only one
ter
region was observed at the time of sporulation division. A second
ter
region, indicative of chromosome separation, was not distinguishable until engulfment was nearing completion, when one was in the mother cell and the other in the prespore. Separation of the two
ter
regions depended on the DNA translocase SpoIIIE. It is concluded that SpoIIIE is required during spore formation for chromosome separation as well as for translocation; SpoIIIE is not required for separation during vegetative growth.
Spindle proteins Aurora A and BUB1B, but not Mad2, are aberrantly expressed in dysplastic mucosa of patients with longstanding ulcerative colitis
Authors: Burum-Auensen E , DeAngelis P M , Schjølberg A R , Røislien Jo , Andersen S N , Clausen O P F
Published: February 2007

Background:

Long term ulcerative colitis (UC) increases the risk of colorectal cancer (CRC). DNA aneuploidy is a common feature of both dysplastic and non-dysplastic colonic epithelia from patients with longstanding UC, and is regarded as an early sign of possible malignant transformation. The spindle proteins Aurora A, BUB1B and Mad2 have been implicated as contributors to aneuploidy and carcinogenesis.

Aims:

To investigate the role of these spindle proteins in relation to DNA aneuploidy and during the progressive morphological changes in ulcerative colitis associated colorectal cancer (UCCRC).

Methods:

Tissue microarrays were made from 31 colectomy specimens from patients with longstanding UC. Expression of Aurora A, BUB1B and Mad2 was investigated by immunohistochemistry and their relation to ploidy status, mucosal morphology and Ki67 levels was explored.

Results:

Expression of Aurora A and BUB1B was significantly associated with the progressive morphological changes of UCCRC. In the progression from non-dysplastic to dysplastic mucosa, Aurora A expression decreased while BUB1B expression increased. There was an increasing incidence of aneuploidy with progression towards cancer; expression of all spindle proteins was associated with the level of Ki67 but not with aneuploidy.

Conclusion:

Due to the significant differences in Aurora A and BUB1B expression in dysplastic compared non-dysplastic mucosa, these proteins may serve as putative biological markers for the progressive morphological changes in UC associated carcinogenesis. The close relationship to Ki67 levels reflect that spindle proteins are expressed in tissues with a high proliferative rate; a role for these proteins in the development of aneuploidy was not found.
HER2 testing in the UK: consensus from a national consultation
Authors: Dowsett Mitch , Hanby A M , Laing R , Walker R
Published: February 2007

Objective:To gain an understanding of current attitudes among oncologists and pathologists to prospective HER2 testing in breast cancer and to gauge whether a national consensus exists regarding extent and quality of testing.

Design:Qualitative study, with semi-quantitative components, using emailed questionnaires and open-ended discussion documents.

Participants:186 relevant specialists, including 76 breast oncologists and 99 pathologists, representing all but three of the UK cancer networks.

Results:A strong consensus was seen in favour of universal, non-selective testing for HER2 at the point of breast cancer diagnosis. Similarly, an overwhelming majority of participants agreed that, to optimise the quality of test results, all laboratories undertaking HER2 testing should be CPA-accredited, participate in the recognised national external quality assessment scheme (UK NEQAS), and carry out a formal annual audit of its testing service. A further recommendation that testing be restricted to laboratories undertaking a minimum 250 tests per annum for immunohistochemistry and 100 tests per annum for in situ hybridisation techniques met with majority support. However, this was not a clear consensus; a significant minority of participants favoured continued use of local services falling short of these criteria.

Conclusion:This study was successful in gauging national specialist opinion regarding the extent and quality assurance of HER2 testing in the UK.
Benefits, risks and ethical considerations in translation of stem cell research to clinical applications in Parkinson’s disease
Authors: Master Zubin , McLeod Marcus , Mendez Ivar
Published: February 2007

Stem cells are likely to be used as an alternate source of biological material for neural transplantation to treat Parkinson’s disease in the not too distant future. Among the several ethical criteria that must be fulfilled before proceeding with clinical research, a favourable benefit to risk ratio must be obtained. The potential benefits to the participant and to society are evaluated relative to the risks in an attempt to offer the participants a reasonable choice. Through examination of preclinical studies transplanting stem cells in animals and the transplantation of fetal tissue in patients with Parkinson’s disease, a current set of potential benefits and risks for neural transplantation of stem cells in clinical research of Parkinson’s disease are derived. The potential benefits to research participants undergoing stem cell transplantation are relief of parkinsonian symptoms and decreasing doses of parkinsonian drugs. Transplantation of stem cells as a treatment for Parkinson’s disease may benefit society by providing knowledge that can be used to help determine better treatments in the future. The risks to research participants undergoing stem cell transplantation include tumour formation, inappropriate stem cell migration, immune rejection of transplanted stem cells, haemorrhage during neurosurgery and postoperative infection. Although some of these risks are general to neurosurgical transplantation and may not be reduced for participants, the potential risk of tumour formation and inappropriate stem cell migration must be minimised before obtaining a favourable potential benefit to risk calculus and to provide participants with a reasonable choice before they enrol in clinical studies.
Rasing the ivory tower: the production of knowledge and distrust of medicine among African Americans
Authors: Wasserman J , Flannery M A , Clair J M
Published: February 2007

African American distrust of medicine has consequences for treatment seeking and healthcare behaviour. Much work has been done to examine acute events (eg, Tuskegee Syphilis Study) that have contributed to this phenomenon and a sophisticated bioethics discipline keeps watch on current practices by medicine. But physicians and clinicians are not the only actors in the medical arena, particularly when it comes to health beliefs and distrust of medicine. The purpose of this paper is to call attention not just to ethical shortcomings of the past, but to the structural contexts of those events and the contributions and responsibilities of popular media and academic disciplines in the production of (often mythic) knowledge. We argue that ignoring context and producing inaccurate work has real impacts on health and healthcare, particularly for African Americans, and thus engenders ethical obligations incumbent on disciplines traditionally recognised as purely academic.
Overexpression of cysteine dioxygenase reduces intracellular cysteine and glutathione pools in HepG2/C3A cells
Authors: Dominy John E. , Hwang Jesse , Stipanuk Martha H.
Published: February 2007

Cysteine levels are carefully regulated in mammals to balance metabolic needs against the potential for cytotoxicity. It has been postulated that one of the major regulators of intracellular cysteine levels in mammals is cysteine dioxygenase (CDO). Hepatic expression of this catabolic enzyme increases dramatically in response to increased cysteine availability and may therefore be part of a homeostatic response to shunt excess toxic cysteine to more benign metabolites such as sulfate or taurine. Direct experimental evidence, however, is lacking to support the hypothesis that CDO is capable of altering steady-state intracellular cysteine levels. In this study, we expressed either the wild-type (WT) or a catalytically inactivated mutant (H86A) isoform of CDO in HepG2/C3A cells (which do not express endogenous CDO protein) and cultured them in different concentrations of extracellular cysteine. WT CDO, but not H86A CDO, was capable of reducing intracellular cysteine levels in cells incubated in physiologically relevant concentrations of cysteine. WT CDO also decreased the glutathione pool and potentiated the toxicity of CdCl2. These results demonstrate that CDO is capable of altering intracellular cysteine levels as well as glutathione levels.
Sex-specific effects of placental restriction on components of the metabolic syndrome in young adult sheep
Authors: Owens J. A. , Thavaneswaran P. , De Blasio M. J. , McMillen I. C. , Robinson J. S. , Gatford K. L.
Published: February 2007

Prenatal and early postnatal life experiences, reflected by size at birth and postnatal catch-up growth, contribute to the risk of developing the metabolic syndrome in adulthood, but their relative importance is unclear. Therefore, we determined the effects of restricted placental and fetal growth on components of the metabolic syndrome in young adult sheep and the relationships of the latter to size at birth and early postnatal growth. Fasting plasma metabolites, glucose tolerance (by intravenous glucose tolerance test, IVGTT), insulin secretion and sensitivity, and resting blood pressure were measured in 22 control and 20 placentally restricted (PR) 1-yr-old sheep. In male sheep, PR increased the initial rise in glucose during an IVGTT and reduced diastolic blood pressure, and small size at birth independently predicted reduced adult size, glucose tolerance, and fasting plasma insulin and insulin disposition of glucose metabolism but increased insulin disposition of circulating FFAs. Also in males, high fractional growth rates in early postnatal life independently predicted impaired early glucose clearance during an IVGTT. In female animals, PR increased insulin sensitivity of glucose metabolism and reduced fasting plasma FFAs, and thinness at birth predicted increased adult size, fasting blood glucose, and pulse pressure. In conclusion, PR and small size at birth are associated with more components of the metabolic syndrome in adult male than in adult female sheep, with few independent effects of early postnatal growth. These sex differences in the onset and extent of adverse metabolic consequences after prenatal restraint in the sheep are consistent with observations in humans.
Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase
Authors: Saini Harjot K. , Dhalla Naranjan S.
Published: February 2007

Although inhibition of the sarcolemmal (SL) Na+-K+-ATPase is known to cause an increase in the intracellular concentration of Ca2+([Ca2+]i) by stimulating the SL Na+/Ca2+exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca2+]iis not fully understood. Isolated rat cardiomyocytes were treated with or without different agents that modify Ca2+movements by affecting various SL sites and were then exposed to ouabain. Ouabain was observed to increase the basal levels of both [Ca2+]iand intracellular Na+concentration ([Na+]i) as well as to augment the KCl-induced increases in both [Ca2+]iand [Na+]iin a concentration-dependent manner. The ouabain-induced changes in [Na+]iand [Ca2+]iwere attenuated by treatment with inhibitors of SL Na+/H+exchanger and SL Na+channels. Both the ouabain-induced increase in basal [Ca2+]iand augmentation of the KCl response were markedly decreased when cardiomyocytes were exposed to 0–10 mM Na+. Inhibitors of SL NCX depressed but decreasing extracellular Na+from 105–35 mM augmented the ouabain-induced increase in basal [Ca2+]iand the KCl response. Not only was the increase in [Ca2+]iby ouabain dependent on the extracellular Ca2+concentration, but it was also attenuated by inhibitors of SL L-type Ca2+channels and store-operated Ca2+channels (SOC). Unlike the SL L-type Ca2+-channel blocker, the blockers of SL Na+channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca2+]i. These results support the view that in addition to SL NCX, SL L-type Ca2+channels and SL SOC may be involved in raising [Ca2+]ion inhibition of the SL Na+-K+-ATPase by ouabain. Furthermore, both SL Na+/H+exchanger and Na+channels play a critical role in the ouabain-induced Ca2+increase in cardiomyocytes.
Efficient transgenic rat production by a lentiviral vector
Authors: Michalkiewicz Mieczyslaw , Michalkiewicz Teresa , Geurts Aron M. , Roman Richard J. , Slocum Glenn R. , Singer Oded , Weihrauch Dorothee , Greene Andrew S. , Kaldunski Mary , Verma Inder M. , Jacob Howard J. , Cowley Allen W.
Published: February 2007

A lentiviral construct for an enhanced green fluorescent protein (eGFP) driven by a chicken β-actin promoter, cytomegalovirus enhancer, and intronic sequences from rabbit β-globin (CAG) was used to produce transgenic lines of rats for evaluation of the usefulness of this approach in gene function studies. Fertilized eggs were collected from inbred Dahl S and outbred Sprague-Dawley rats, and ∼100 pl of concentrated virus were microinjected into the perivitrelline space of one-cell embryos. Of 121 embryos injected, 60 pups (49.6%) were born. Transgenic rates averaged 22% in Dahl S and 14% in Sprague-Dawley rats. Copy number ranged from one to four in the founders, and the inheritance of the transgene in a subsequent F1population was 48.2%. The small number of insertion sites enabled us to derive inbred transgenic lines with a single copy of the transgene within one generation. Sequencing of each transgene insertion site revealed that they inserted as single copies with a preference for the introns of genes. The CAG promoter drove high levels of eGFP expression in brain, kidney, heart, and vasculature, making it very suitable for exploring the cardiovascular function of newly discovered genes. The pattern of eGFP expression was similar across five different F1transgenic lines, indicating that the expression of the transgene was independent of its chromosomal position. Thus lentiviral transgenesis provides a powerful tool for the production of transgenic inbred rats and will enhance the usefulness of this species in gene discovery and target validation studies.
Relationship between tissue Doppler-derived RV systolic function and invasive hemodynamic measurements
Authors: Hori Yasutomo , Kano Takuto , Hoshi Fumio , Higuchi Sei-ichi
Published: February 2007

Tissue Doppler imaging (TDI) is effective in assessing right ventricular (RV) function, but the relationship between invasive measurements and RV-TDI remains unclear. We investigated the RV systolic function by using the TDI-derived systolic myocardial (Sa) velocity and myocardial performance index (MPI). Beagles ( n = 7) were anesthetized in the right lateral recumbent position. A 3.5-Fr micromanometer-tipped catheter was placed in the RV to determine the hemodynamic changes. Dobutamine (5.0 and 10 μg·kg−1·min−1) and esmolol (50 and 100 μg·kg−1·min−1) were infused intravenously. Pulsed Doppler (PD) and TDI measurements were performed in the apical four-chamber view. Compared with baseline, the PD-MPI decreased significantly with the dobutamine infusion at 5 μg·kg−1·min−1 ( P < 0.05). Both dobutamine infusions significantly decreased the TDI-MPI ( P < 0.01, P < 0.05). Esmolol increased the PD- and TDI-MPI but not significantly. Dobutamine significantly increased the Sa velocity (both P < 0.001), whereas esmolol had no effect. The Sa velocity was strongly correlated with the peak positive derivative of the RV pressure (+dP/dt; r = 0.93). The negative correlation between the +dP/dt and TDI-MPI ( r = −086) was greater that with the PD-MPI ( r = −0.54). Stepwise regression analysis showed that the Sa velocity and PD-derived isovolumic contraction time were identified to predict the +dP/dt ( r = 0.94, r2 = 0.89; P < 0.001). We determined that the systolic myocardial velocity and TDI-MPI were strongly correlated with the RV contractility. These results suggest that the TDI-derived systolic myocardial velocity and MPI predict RV systolic function.
Preemptive heme oxygenase-1 gene delivery reveals reduced mortality and preservation of left ventricular function 1 yr after acute myocardial infarction
Authors: Liu Xiaoli , Simpson Jeremy A. , Brunt Keith R. , Ward Christopher A. , Hall Sean R. R. , Kinobe Robert T. , Barrette Valerie , Tse M. Yat , Pang Stephen C. , Pachori Alok S. , Dzau Victor J. , Ogunyankin Kofo O. , Melo Luis G.
Published: February 2007

We reported previously that predelivery of heme oxygenase-1 (HO-1) gene to the heart by adeno-associated virus-2 (AAV-2) markedly reduces ischemia and reperfusion (I/R)-induced myocardial injury. However, the effect of preemptive HO-1 gene delivery on long-term survival and prevention of postinfarction heart failure has not been determined. We assessed the effect of HO-1 gene delivery on long-term survival, myocardial function, and left ventricular (LV) remodeling 1 yr after myocardial infarction (MI) using echocardiographic imaging, pressure-volume (PV) analysis, and histomorphometric approaches. Two groups of Lewis rats were injected with 2 × 1011 particles of AAV-LacZ (control) or AAV-human HO-1 (hHO-1) in the anterior-posterior apical region of the LV wall. Six weeks after gene transfer, animals were subjected to 30 min of ischemia by ligation of the left anterior descending artery followed by reperfusion. Echocardiographic measurements and PV analysis of LV function were obtained at 2 wk and 12 mo after I/R. One year after acute MI, mortality was markedly reduced in the HO-1-treated animals compared with the LacZ-treated animals. PV analysis demonstrated significantly enhanced LV developed pressure, elevated maximal dP/d t, and lower end-diastolic volume in the HO-1 animals compared with the LacZ animals. Echocardiography showed a larger apical anterior-to-posterior wall ratio in HO-1 animals compared with LacZ animals. Morphometric analysis revealed extensive myocardial scarring and fibrosis in the infarcted LV area of LacZ animals, which was reduced by 62% in HO-1 animals. These results suggest that preemptive HO-1 gene delivery may be useful as a therapeutic strategy to reduce post-MI LV remodeling and heart failure.
CytochromeP-450 epoxygenases protect endothelial cells from apoptosis induced by tumor necrosis factor-α via MAPK and PI3K/Akt signaling pathways
Authors: Yang Shilin , Lin Li , Chen Ji-Xiong , Lee Craig R. , Seubert John M. , Wang Yan , Wang Hong , Chao Zhong-Ren , Tao De-Ding , Gong Jian-Ping , Lu Zai-Ying , Wang Dao Wen , Zeldin Darryl C.
Published: February 2007

Endothelial cells play a vital role in the maintenance of cardiovascular homeostasis. Epoxyeicosatrienoic acids (EETs), cytochrome P-450 (CYP) epoxygenase metabolites of arachidonic acid in endothelial cells, possess potent and diverse biological effects within the vasculature. We evaluated the effects of overexpression of CYP epoxygenases on tumor necrosis factor-α (TNF-α)-induced apoptosis in bovine aortic endothelial cells. CYP epoxygenase overexpression significantly increased endothelial cell viability and inhibited TNF-α induction of endothelial cell apoptosis as evaluated by morphological analysis of nuclear condensation, DNA laddering, and fluorescent-activated cell sorting (FACS) analysis. CYP epoxygenase overexpression also significantly inhibited caspase-3 activity and downregulation of Bcl-2 expression induced by TNF-α. The antiapoptotic effects of CYP epoxygenase overexpression were significantly attenuated by inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK signaling pathways; however, inhibition of endothelial nitric oxide synthase activity had no effect. Furthermore, CYP epoxygenase overexpression significantly attenuated the extent of TNF-α-induced ERK1/2 dephosphorylation in a time-dependent manner and significantly increased PI3K expression and Akt phosphorylation in both the presence and absence of TNF-α. Collectively, these results suggest that CYP epoxygenase overexpression, which is known to increase EET biosynthesis, significantly protects endothelial cells from apoptosis induced by TNF-α. This effect is mediated, at least in part, through inhibition of ERK dephosphorylation and activation of PI3K/Akt signaling.
Restoration of impaired endothelium-dependent coronary vasodilation in failing heart: role of eNOS phosphorylation and CGMP/cGK-I signaling
Authors: Gill Robert M. , Braz Julian C. , Jin Najia , Etgen Gary J. , Shen Weiqun
Published: February 2007

In congestive heart failure (CHF), coronary vascular relaxation is associated with endothelial dysfunction and nitric oxide (NO) deficiency. This study explored the reversibility of this process in hearts recovering from CHF and its related mechanisms. Dogs were chronically instrumented to measure cardiac function and coronary blood flow (CBF). Heart failure was induced by right ventricular pacing at 240 beats/min for 3–4 wk, and cardiac recovery (CR) was allowed by the termination of cardiac pacing for 3–4 wk after the development of CHF, in which left ventricular contractile function was restored by 80–90%. The endothelium-dependent CBF response to bradykinin and acetylcholine was depressed in CHF and fully restored in CR. Myocardial NOx (nitrate/nitrite), endothelial NO synthase (eNOS) mRNA expression, total protein, and phosphorylated eNOS decreased significantly in failing hearts. However, myocardial NOx recovered to 78% of control and phosphorylated eNOS was fully restored in CR, despite the fact that eNOS mRNA expression and protein levels remained lower than control. Furthermore, the endothelium-independent CBF response to nitroglycerin did not change in CHF; however, it increased by 75% in CR, in conjunction with a near threefold increase in the phosphorylation of vasodilation-stimulated phosphoprotein (VASP) at Ser239in recovering hearts. Thus the complete restoration of endothelium-dependent coronary vascular relaxation during cardiac recovery from CHF was mediated by 1) a restoration of phosphorylated eNOS for partial recovery of the NO production and 2) an increase in cGMP/cGMP-dependent protein kinase-I pathway signaling activity for the enhancement of coronary vascular smooth muscle relaxation in response to NO.
Anti-atherosclerotic effects of sirolimus on human vascular smooth muscle cells
Authors: Ma Kun L. , Ruan Xiong Z. , Powis Stephen H. , Moorhead John F. , Varghese Zac
Published: February 2007

Sirolimus is a potent immunosuppressive agent and has an anti-atherosclerotic effect through its anti-proliferative property. The present study was undertaken to investigate the effect of sirolimus on intracellular cholesterol homeostasis in human vascular smooth muscle cells (VSMCs) in the presence of inflammatory cytokine IL-1β. We explored the effect of sirolimus on the lipid accumulation of VSMCs in the presence of IL-1β, using Oil Red O staining and quantitative measurement of intracellular cholesterol. The effect of sirolimus on the gene and protein expression of lipoprotein receptors and ATP binding cassettes (ABCA1 and ABCG1) was examined by real-time PCR and Western blotting, respectively. Furthermore, the effect of sirolimus on cholesterol efflux from VSMCs in the presence or absence of IL-1β was also investigated using [3H] cholesterol efflux. Finally, we examined the effect of sirolimus on the production of inflammatory cytokines in VSMCs using ELISA. Sirolimus reduced intracellular lipid accumulation in VSMCs mediated by IL-1β possibly due to the reduction of expression of low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) receptors. Sirolimus increased cholesterol efflux from VSMCs and overrode the suppression of cholesterol efflux induced by IL-1β. Sirolimus also increased ABCA1 and ABCG1 genes expression, even in the presence of IL-1β. We further confirmed that sirolimus inhibited mRNA and protein expression of inflammatory cytokines IL-6, tumor necrosis factor-α, IL-8, and monocyte chemoattractant protein-1. Inhibition of lipid uptake together with increasing cholesterol efflux and the inhibition of inflammatory cytokines are all important aspects of the anti-atherosclerotic effects of sirolimus on VSMCs.
In vitro study of LDL transport under pressurized (convective) conditions
Authors: Cancel Limary M. , Fitting Andrew , Tarbell John M.
Published: February 2007

It is difficult to assess the transport pathways that carry low-density lipoprotein (LDL) into the artery wall in vivo, and there has been no previous in vitro study that has examined transendothelial transport under physiologically relevant pressurized (convective) conditions. Therefore, we measured water, albumin, and LDL fluxes across bovine aortic endothelial cell (BAEC) monolayers in vitro and determined the relative contributions of vesicles, paracellular transport through “breaks” in the tight junction, and “leaky” junctions associated with dying or dividing cells. Our results show that leaky junctions are the dominant pathway for LDL transport (>90%) under convective conditions and that albumin also has a significant component of transport through leaky junctions (44%). Transcellular transport of LDL by receptor-mediated processes makes a minor contribution (<10%) to overall transport under convective conditions.
High-resolution imaging of murine myocardial infarction with delayed-enhancement cine micro-CT
Authors: Nahrendorf Matthias , Badea Cristian , Hedlund Laurence W. , Figueiredo Jose-Luiz , Sosnovik David E. , Johnson G. Allan , Weissleder Ralph
Published: February 2007

The objective of this study was to determine the feasibility of delayed-enhancement micro-computed tomography (μCT) imaging to quantify myocardial infarct size in experimental mouse models. A total of 20 mice were imaged 5 or 35 days after surgical ligation of the left coronary artery or sham surgery ( n = 6 or 7 per group). We utilized a prototype μCT that covers a three-dimensional (3D) volume with an isotropic spatial resolution of 100 μm. A series of image acquisitions were started after a 200 μl bolus of a high-molecular-weight blood pool CT agent to outline the ventricles. CT imaging was continuously performed over 60 min, while an intravenous constant infusion with iopamidol 370 was started at a dosage of 1 ml/h. Thirty minutes after the initiation of this infusion, signal intensity in Hounsfield units was significantly higher in the infarct than in the remote, uninjured myocardium. Cardiac morphology and motion were visualized with excellent contrast and in fine detail. In vivo CT determination of infarct size at the midventricular level was in good agreement with ex vivo staining with triphenyltetrazolium chloride [5 days post-myocardial infarction (MI): r2= 0.86, P < 0.01; 35 days post-MI: r2= 0.92, P < 0.01]. In addition, we detected significant left ventricular remodeling consisting of left ventricular dilation and decreased ejection fraction. 3D cine μCT reliably and rapidly quantifies infarct size and assesses murine anatomy and physiology after coronary ligation, despite the small size and fast movement of the mouse heart. This efficient imaging tool is a valuable addition to the current phenotyping armamentarium and will allow rapid testing of novel drugs and cell-based interventions in murine models.
Increased sympathetic venoconstriction and reactivity to norepinephrine in mesenteric veins in anesthetized DOCA-salt hypertensive rats
Authors: Xu Hui , Fink Gregory D. , Galligan James J.
Published: February 2007

Increased sympathetic nervous activity (SNA) elevates venomotor tone in deoxycorticosterone acetate (DOCA)-salt hypertension. We studied the mechanisms by which the SNA increases venomotor tone in DOCA-salt hypertension by making in situ intracellular recordings of venous smooth muscle cell (VSMC) membrane potential ( Em) and measurement of outside diameter (OD) in mesenteric veins (MV) and mesenteric arteries (MA) of anesthetized rats. We also studied norepinephrine (NE)- and endothelin-1 (ET-1)-induced increases in MA or MV perfusion pressure (PP) in vitro. Em in DOCA-salt MV was depolarized compared with sham MV. Prazosin hyperpolarized VSMC Em in DOCA-salt but not in sham MV. NE concentration-response curves (CRCs) for OD decreases in MV from DOCA-salt rats were left-shifted with an increased maximum response ( Emax) compared with sham MV. NE CRCs for OD decreases in MA were right-shifted with reduced Emax in DOCA-salt compared with sham rats. ET-1 CRCs were similar in DOCA-salt and sham MV but were right-shifted with reduced Emax in DOCA-salt MA. NE CRCs for MAPP increases were left-shifted without a change in Emax in DOCA-salt rats. NE did not change MVPP. MAPP and MVPP for ET-1 CRCs were similar in sham and DOCA-salt rats, but Emax for MAPP was reduced in DOCA-salt rats. Hematoxylin staining revealed hypertrophy in DOCA-salt MA but not in MV. We conclude that there is increased reactivity to NE released from the sympathetic nervous system in DOCA-salt MV that causes VSMC depolarization and increased venomotor tone. In DOCA-salt rats, in vivo ET-1 reactivity is maintained in MV, but reduced in MA.
Basolateral Cl channels in primary airway epithelial cultures
Authors: Fischer Horst , Illek Beate , Finkbeiner Walter E. , Widdicombe Jonathan H.
Published: February 2007

Salt and water absorption and secretion across the airway epithelium are important for maintaining the thin film of liquid lining the surface of the airway epithelium. Movement of Cl across the apical membrane involves the CFTR Cl channel; however, conductive pathways for Cl movement across the basolateral membrane have been little studied. Here, we determined the regulation and single-channel properties of the Cl conductance ( GCl) in airway surface epithelia using epithelial cultures from human or bovine trachea and freshly isolated ciliated cells from the human nasal epithelium. In Ussing chamber studies, a swelling-activated basolateral GCl was found, which was further stimulated by forskolin and blocked by N-phenylanthranilic acid (DPC) = sucrose > flufenamic acid = niflumic acid = glibenclamide > CdCl2 = 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) = DIDS = ZnCl2 > tamoxifen > 4,4′-dinitro-2,2′-stilbene-disulfonate disodium salt (DNDS). In whole cell patch-clamp experiments, three types of GCl were identified: 1) a voltage-activated, DIDS- (but not Cd-) blockable and osmosensitive GCl; 2) an inwardly rectifying, hyperpolarization-activated and Cd-sensitive GCl; and 3) a forskolin-activated, linear GCl, which was insensitive to Cd and DIDS. In cell-attached patch-clamp recordings, the basolateral pole of isolated ciliated cells expressed three types of Cl channels: 1) an outwardly rectifying, swelling-activated Cl channel; 2) a strongly inwardly rectifying Cl channel; and 3) a forskolin-activated, low-conductance channel. We propose that, depending on the driving force for Cl across the apical membrane, basolateral Cl channels confine Cl− secretion or support transcellular Cl− absorption.
Modulation of pulmonary vascular smooth muscle cell phenotype in hypoxia: role of cGMP-dependent protein kinase
Authors: Zhou Weilin , Dasgupta Chiranjib , Negash Sewite , Raj J. Usha
Published: February 2007

Chronic hypoxia triggers pulmonary vascular remodeling, which is associated with a modulation of the vascular smooth muscle cell (SMC) phenotype from a contractile, differentiated to a synthetic, dedifferentiated state. We previously reported that acute hypoxia represses cGMP-dependent protein kinase (PKG) expression in ovine fetal pulmonary venous SMCs (FPVSMCs). Therefore, we tested if altered expression of PKG could explain SMC phenotype modulation after exposure to hypoxia. Hypoxia-induced reduction in PKG protein expression strongly correlated with the repressed expression of SMC phenotype markers, myosin heavy chain (MHC), calponin, vimentin, α-smooth muscle actin (αSMA), and thrombospondin (TSP), indicating that hypoxic exposure of SMC induced phenotype modulation to dedifferentiated state, and PKG may be involved in SMC phenotype modulation. PKG-specific small interfering RNA (siRNA) transfection in FPVSMCs significantly attenuated calponin, vimentin, and MHC expression, with no effect on αSMA and TSP. Treatment with 30 μM Drosophila Antennapedia (DT-3), a membrane-permeable peptide inhibitor of PKG, attenuated the expression of TSP, MHC, αSMA, vimentin, and calponin. The results from PKG siRNA and DT-3 studies indicate that hypoxia-induced reduction in protein expression was also similarly impacted by PKG inhibition. Overexpression of PKG in FPVSMCs by transfection with a full-length PKG construct tagged with green fluorescent fusion protein (PKG-GFP) reversed the effect of hypoxia on the expression of SMC phenotype marker proteins. These results suggest that PKG could be one of the determinants for the expression of SMC phenotype marker proteins and may be involved in the maintenance of the differentiated phenotype in pulmonary vascular SMCs in hypoxia.
Activation of HGF/c-Met pathway contributes to the reactive oxygen species generation and motility of small cell lung cancer cells
Authors: Jagadeeswaran Ramasamy , Jagadeeswaran Sujatha , Bindokas Vytas P. , Salgia Ravi
Published: February 2007

Small cell lung cancer (SCLC) is a difficult disease to treat and sometimes has overexpression or mutation of c-Met receptor tyrosine kinase. The effects of c-Met/hepatocyte growth factor (c-Met/HGF, ligand for c-Met) on activation of reactive oxygen species (ROS) was determined. HGF stimulation of c-Met-overexpressing H69 SCLC cells (40 ng/ml, 15 min) resulted in an increase of ROS, measured with fluorescent probe 2′-7′-dichlorofluorescein diacetate (DCFH-DA) or dihydroethidine (DHE) but not in c-Met-null H446 cells. ROS was increased in juxtamembrane (JM)-mutated variants (R988C and T1010I) of c-Met compared with wild-type c-Met-expressing cells. ROS was significantly inhibited by preincubation of SCLC cells with pyrrolidine dithiocarbamate (PDTC, 100 μM) and/or SU11274 (small molecule c-Met tyrosine kinase inhibitor, 2 μM) for 3 h. PDTC and SU11274 also abrogated the HGF proliferative signal and cell motility in a cooperative fashion. H2O2 treatment of SCLC cells (over 15 min) led to phosphorylation of c-Met receptor tyrosine kinase and further upregulated downstream phosphorylation of phospho-AKT, ERK1/2, and paxillin in a dose-dependent manner (125 μM to 500 μM). c-Met is an important target in lung cancer, and the pathways responsible for ROS generation together may provide novel therapeutic intervention.
Interaction of interleukin-6 and the BMP pathway in pulmonary smooth muscle
Authors: Hagen Moira , Fagan Karen , Steudel Wolfgang , Carr Michelle , Lane Kirk , Rodman David M. , West James
Published: February 2007

The majority of familial pulmonary arterial hypertension (PAH) cases are caused by mutations in the type 2 bone morphogenetic protein receptor (BMPR2). However, less than one-half of BMPR2 mutation carriers develop PAH, suggesting that the most important function of BMPR2 mutation is to cause susceptibility to a “second hit.” There is substantial evidence from the literature implicating dysregulated inflammation, in particular the cytokine IL-6, in the development of PAH. We thus hypothesized that the BMP pathway regulates IL-6 in pulmonary tissues and conversely that IL-6 regulates the BMP pathway. We tested this in vivo using transgenic mice expressing an inducible dominant negative BMPR2 in smooth muscle, using mice injected with an IL-6-expressing virus, and in vitro using small interfering RNA (siRNA) to BMPR2 in human pulmonary artery smooth muscle cells (PA SMC). Consistent with our hypothesis, we found upregulation of IL-6 in both the transgenic mice and in cultured PA SMC with siRNA to BMPR2; this could be abolished with p38MAPKinhibitors. We also found that IL-6 in vivo caused a twofold increase in expression of the BMP signaling target Id1 and caused increased BMP activity in a luciferase-reporter assay in PA SMC. Thus we have shown both in vitro and in vivo a complete negative feedback loop between IL-6 and BMP, suggesting that an important consequence of BMPR2 mutations may be poor regulation of cytokines and thus vulnerability to an inflammatory second hit.