bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutational Analysis of Hemoglobin Binding and Heme Utilization by a Bacterial Hemoglobin Receptor
Authors: Fusco W. G. , Choudhary N. R. , Council S. E. , Collins E. J. , Leduc I.
Published: May 2013

Iron is an essential nutrient for most living organisms. To acquire iron from their environment, Gram-negative bacteria use TonB-dependent transporters that bind host proteins at the bacterial surface and transport iron or heme to the periplasm via the Ton machinery. TonB-dependent transporters are barrel-shaped outer membrane proteins with 22 transmembrane domains, 11 surface-exposed loops, and a plug domain that occludes the pore. To identify key residues of TonB-dependent transporters involved in hemoglobin binding and heme transport and thereby locate putative protective epitopes, the hemoglobin receptor of
Haemophilus ducreyi
HgbA was used as a model of iron/heme acquisition from hemoglobin. Although all extracellular loops of HgbA are required by
H. ducreyi
to use hemoglobin as a source of iron/heme, we previously demonstrated that hemoglobin binding by HgbA only involves loops 5 and 7. Using deletion, substitution, and site-directed mutagenesis, we were able to differentiate hemoglobin binding and heme acquisition by HgbA. Deletion or substitution of the GYEAYNRQWWA region of loop 5 and alanine replacement of selected histidines affected hemoglobin binding by HgbA. Conversely, mutation of the phenylalanine in the loop 7 FRAP domain or substitution of the NRQWWA motif of loop 5 significantly abrogated utilization of heme from hemoglobin. Our findings show that hemoglobin binding and heme utilization by a bacterial hemoglobin receptor involve specific motifs of HgbA.
Characterization of McuB, a Periplasmic Chaperone-Like Protein Involved in the Assembly of Myxococcus Spore Coat
Authors: Zhu Wei , Wu Miaomiao , Cao Shanshan , Peng Yongxing , Mao Xiaohua
Published: May 2013

The MXAN3885 to -3882 gene locus cluster (designated here
mcuABCD
) of
Myxococcus xanthus
encodes a member of the archaic chaperone-usher (CU) systems that functions in spore coat formation. We show here that McuD, a putative spore coat protein, affects cellular accumulation and cell surface localization of the spore coat protein McuA. We previously reported that genetic disruption of the putative usher McuC nearly eliminates surface display of McuA and show here that lack of the periplasmic chaperone-like protein McuB, which forms a complex with McuA, has a similar effect. Deletion mutation confirms that the G1 β strand of McuB is absolutely essential for the stability and secretion of McuA. Site-directed mutagenesis identified two additional alternating hydrophobic residues Ile113 and Val115, together with the highly conserved proline within the G1 strand, as critical residues for chaperone function. These findings suggest that the assembly proteins McuB and McuC mediate the transport of McuA onto the cell surface and that McuA may interact with another spore coat protein, McuD, for its secretion. Importantly, although our data argue that the
M. xanthus
CU system is likely to use the basic principle of donor strand complementation (DSC), as in the cases of classical CU pathways, to promote folding and stabilization of the structural subunit(s), the periplasmic chaperone McuB appears to exhibit structural variation in mediating chaperone-subunit interaction.
Acupuncture in Preventing Postoperative Anaesthesia-Related Sore Throat: A Comparison with No Acupuncture
Authors: Esmaeili Sara , Alizadeh Reza , Shoar Saeed , Naderan Mohammad , Shoar Nasrin
Published: May 2013

Background

Postoperative sore throat occurs frequently in surgical patients as a result of tracheal intubation. Despite advances in medical and anaesthetic care, the incidence of postoperative sore throat remains high. Our study aimed to assess the efficacy of acupuncture in the prevention of postoperative sore throat following general anaesthesia.

Methods

A non-randomised clinical study (NRS) was carried out in a tertiary care hospital in Tehran, Iran on 228 consecutive patients undergoing elective surgeries under general anaesthesia. Of these, 114 patients were assigned to the acupuncture group and treated with body acupuncture at the PC6 point, while the remaining 114 patients received no preventive care and were treated with routine medical treatment where necessary. The incidence of sore throat within the first 24 h postoperatively was then compared between the two study groups.

Results

Of the 114 patients in the acupuncture group, 16 patients (14%) experienced sore throat within the first 24 h postoperatively, which was significantly lower compared to the 34 patients (29.8%) with sore throat in the medical treatment group (p<0.05). However, the frequency of patients with severe sore throat requiring medical treatment did not differ significantly (12.3% vs 16.7%, p>0.05).

Conclusions

Our study suggests that acupuncture could be considered as an option in prevention of postoperative anaesthesia-related sore throat.
Individual variability in compensatory eating following acute exercise in overweight and obese women
Authors: Hopkins Mark , Blundell John E , King Neil A
Published: May 2013

Background

While compensatory eating following acute aerobic exercise is highly variable, little is known about the underlying mechanisms that contribute to the alterations in exercise-induced eating behaviour.

Methods

Overweight and obese women (body mass index=29.6±4.0 kg/m2) performed a bout of cycling individually tailored to expend 400 kcal (EX) or a time-matched no exercise control condition in a randomised, counter-balanced order. 60 min after the cessation of exercise, an ad libitum test meal was provided. Substrate oxidation and subjective appetite ratings were measured during exercise/time-matched rest, and during the period between the cessation of exercise and food consumption.

Results

While ad libitum energy intake (EI) did not differ between EX and the control condition (666.0±203.9 vs 664.6±174.4 kcal, respectively; ns), there was a marked individual variability in compensatory EI. The difference in EI between EX and the control condition ranged from −234.3 to 278.5 kcal. Carbohydrate oxidation during exercise was positively associated with postexercise EI, accounting for 37% of the variance in EI (r=0.57; p=0.02).

Conclusions

These data indicate that the capacity of acute exercise to create a short-term energy deficit in overweight and obese women is highly variable. Furthermore, exercise-induced CHO oxidation can explain a part of the variability in acute exercise-induced compensatory eating. Postexercise compensatory eating could serve as an adaptive response to facilitate the restoration of carbohydrate balance.
Involving patient research partners has a significant impact on outcomes research: a responsive evaluation of the international OMERACT conferences
Authors: de Wit Maarten , Abma Tineke , Koelewijn-van Loon Marije , Collins Sarah , Kirwan John
Published: May 2013

Objective

To assess the inclusion of patients as international research partners in Outcome Measures in Rheumatology (OMERACT) conferences and how this has influenced the scope and conduct of outcomes research in rheumatology.

Design

A thematic content analysis of OMERACT internal documents, publications and conference proceedings, followed by a responsive evaluation including 32 qualitative semistructured interviews.

Setting

The international, biannual research conference OMERACT 10 (Malaysia, 2010).

Participants

Senior researchers (n=10), junior researchers (n=2), representatives of the pharmaceutical industry and regulators (n=2), conference staff (n=2), new patient delegates (n=8) and experienced patient delegates (n=8).

Results

The role of patients evolved over 10 years from a single patient focus group to full participation in all areas of the meeting and inclusion in research group meetings between conferences. Five main categories of impact emerged: widening the research agenda; including patient relevant outcomes in core sets; enhancing patient reported instruments; changing the culture of OMERACT and consequences outside OMERACT. Patient participants identified previously neglected outcome domains such as fatigue, sleep disturbances and flares which prompted collaborative working on new programmes of research. Specific benefits and challenges for patients and professionals were identified, such as personal fulfilment, widening of research interests, difficulties in establishing equal partnerships and concerns about loss of research rigour.

Conclusions

Including patients as partners in OMERACT conferences has widened its focus and adjusted the way of working. It has resulted in new developments in the research agenda and the use of more patient-relevant outcomes in clinical trials. These collaborations have influenced perceptions and beliefs among many patients and researchers, and led to wider patient involvement as partners in research.
Life-course pathways to psychological distress: a cohort study
Authors: von Stumm Sophie , Deary Ian J , Hagger-Johnson Gareth
Published: May 2013

Objectives

Early life factors, like intelligence and socioeconomic status (SES), are associated with health outcomes in adulthood. Fitting comprehensive life-course models, we tested (1) the effect of childhood intelligence and SES, education and adulthood SES on psychological distress at midlife, and (2) compared alternative measurement specifications (reflective and formative) of SES.

Design

Prospective cohort study (the Aberdeen Children of the 1950s).

Setting

Aberdeen, Scotland.

Participants

12 500 live-births (6282 boys) between 1950 and 1956, who were followed up in the years 2001–2003 at age 46–51 with a postal questionnaire achieving a response rate of 64% (7183).

Outcome measures

Psychological distress at age 46–51 (questionnaire).

Results

Childhood intelligence and SES and education had indirect effects on psychological distress at midlife, mediated by adult SES. Adult SES was the only variable to have a significant direct effect on psychological distress at midlife; the effect was stronger in men than in women. Alternative measurement specifications of SES (reflective and formative) resulted in greatly different model parameters and fits.

Conclusions

Even though formative operationalisations of SES are theoretically appropriate, SES is better specified as reflective than as a formative latent variable in the context of life-course modelling.
Reversible khat-induced hepatitis: two case reports and review of the literature
Authors: Jenkins M G , Handslip R , Kumar M , Mahadeva U , Lucas S , Yamamoto T , Wood D M , Wong T , Dargan P I
Published: May 2013

Recreational chewing ofCatha edulis(khat) leaves is part of the ethnic culture of Somali, Yemeni and other East African societies for its stimulant properties. With increasing emigration, khat use has become common in these ethnic groups once they move to other areas such as Europe and the USA; one-third of the UK Somali population report khat use within the last month. Cathinone, the active component of the khat leaves, is controlled under the UK Misuse of Drugs Act, but the use of the khat plant and its leaves remains not subject to control in the UK. There have been several previous reports of acute hepatitis related to chronic use of khat leading to acute liver failure, and resulting in transplantation or death. We report two cases with severe acute khat-related hepatitis that resolved on cessation of khat use initially, but relapsed with further use, reinforcing the importance of permanent khat cessation to prevent progression to liver failure. With reference to the current literature, we also consider the difficult diagnosis of this disorder, then go on to consider the pathophysiology, mechanisms of liver injury and potential future areas of research.
Caffeine and glucose homeostasis during rest and exercise in diabetes mellitus
Authors: Zaharieva Dessi P. , Riddell Michael C.
Published: May 2013

Caffeine is a substance that has been used in our society for generations, primarily for its effects on the central nervous system that causes wakefulness. Caffeine supplementation has become increasingly more popular as an ergogenic aid for athletes and considerable scientific evidence supports its effectiveness. Because of their potential to alter energy metabolism, the effects of coffee and caffeine on glucose metabolism in diabetes have also been studied both epidemiologically and experimentally. Predominantly targeting the adenosine receptors, caffeine causes alterations in glucose homeostasis by decreasing glucose uptake into skeletal muscle, thereby causing elevations in blood glucose concentration. Caffeine intake has also been proposed to increase symptomatic warning signs of hypoglycemia in patients with type 1 diabetes and elevate blood glucose levels in patients with type 2 diabetes. Other effects include potential increases in glucose counterregulatory hormones such as epinephrine, which can also decrease peripheral glucose disposal. Despite these established physiological effects, increased coffee intake has been associated with reduced risk of developing type 2 diabetes in large-scale epidemiological studies. This review paper highlights the known effects of caffeine on glucose homeostasis and diabetes metabolism during rest and exercise.
Conjugate divergent margins: an introduction
Authors: Mohriak Webster U. , Danforth Al , Post Paul J. , Brown David E. , Tari Gabor C. , Nemčok Michal , Sinha Sudipta T.
Published: May 2013

The main objective of this book is to provide a global overview of divergent margins based on geological and geophysical interpretation of sedimentary basins along the South, Central and North Atlantic conjugate margins, from plate tectonics and crustal scales to a more detailed description of stratigraphical and structural elements that are responsible for petroleum plays. These themes are complemented by geodynamic concepts based on physical and numerical models, and by comparisons with present-day embryonic margins, which are succinctly discussed in some papers.

Supplementary material:

Three plate animations of the Atlantic Ocean are available at
www.geolsoc.org.uk/SUP18620
.
Thrombin (PAR-1)-induced proliferation in astrocytes via MAPK involves multiple signaling pathways
Authors: Wang Hong , Ubl Joachim J. , Stricker Rolf , Reiser Georg
Published: May 2013

Protease-activated receptors (PARs), newly identified members of G protein-coupled receptors, are widely distributed in the brain. Thrombin evokes multiple cellular responses in a large variety of cells by activating PAR-1, -3, and -4. In cultured rat astrocytes we investigated the signaling pathway of thrombin- and PAR-activating peptide (PAR-AP)-induced cell proliferation. Our results show that PAR activation stimulates proliferation of astrocytes through the ERK pathway. Thrombin stimulates ERK1/2 phosphorylation in a time- and concentration-dependent manner. This effect can be fully mimicked by a specific PAR-1-AP but only to a small degree by PAR-3-AP and PAR-4-AP. PAR-2-AP can induce a moderate ERK1/2 activation as well. Thrombin-stimulated ERK1/2 activation is mainly mediated by PAR-1 via two branches: 1) the PTX-sensitive G protein/(βγ-subunits)-phosphatidylinositol 3-kinase branch, and 2) the Gq-PLC-(InsP3receptor)/Ca2+-PKC pathway. Thrombin- or PAR-1-AP-induced ERK activation is partially blocked by a selective EGF receptor inhibitor, AG1478. Nevertheless, transphosphorylation of EGF receptor is unlikely for ERK1/2 activation and is certainly not involved in PAR-1-induced proliferation. The metalloproteinase mechanism involving transactivation of the EGF receptor by released heparin-binding EGF was excluded. EGF receptor activation was detected by the receptor autophosphorylation site, tyrosine 1068. Our data suggest that thrombin-induced mitogenic action in astrocytes occurs independently of EGF receptor transphosphorylation.
What is the role of SNARE proteins in membrane fusion?
Authors: Duman Joseph G. , Forte John G.
Published: May 2013

Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins have been at the fore-front of research on biological membrane fusion for some time. The subcellular localization of SNAREs and their ability to form the so-called SNARE complex may be integral to determining the specificity of intracellular fusion (the SNARE hypothesis) and/or serving as the minimal fusion machinery. Both the SNARE hypothesis and the idea of the minimal fusion machinery have been challenged by a number of experimental observations in various model systems, suggesting that SNAREs may have other functions. Considering recent advances in the SNARE literature, it appears that SNAREs may actually function as part of a complex fusion “machine.” Their role in the machinery could be any one or a combination of roles, including establishing tight membrane contact, formation of a scaffolding on which to build the machine, binding of lipid surfaces, and many others. It is also possible that complexations other than the classic SNARE complex participate in membrane fusion.
PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression
Authors: Yu Ying , Sweeney Michele , Zhang Shen , Platoshyn Oleksandr , Landsberg Judd , Rothman Abraham , Yuan Jason X.-J.
Published: May 2013

Capacitative Ca2+ entry (CCE) through store-operated Ca2+ (SOC) channels plays an important role in returning Ca2+ to the sarcoplasmic reticulum (SR) and regulating cytosolic free Ca2+concentration ([Ca2+]cyt). A rise in [Ca2+]cyt and sufficient Ca2+ in the SR are required for pulmonary artery smooth muscle cell (PASMC) proliferation. We tested the hypothesis that platelet-derived growth factor (PDGF)-mediated PASMC growth involves upregulation of c-Jun and TRPC6, a transient receptor potential cation channel. In rat PASMC, PDGF (10 ng/ml for 0.5–48 h) phosphorylated signal transducer and activator of transcription (STAT3), increased mRNA and protein levels of c-Jun, and stimulated cell proliferation. PDGF treatment also upregulated TRPC6 expression and augmented CCE, elicited by passive depletion of Ca2+ from the SR using cyclopiazonic acid. Furthermore, overexpression of c-Jun stimulated TRPC6 expression and CCE amplitude in PASMC. Downregulation of TRPC6 using an antisense oligonucleotide specifically for human TRPC6 decreased CCE and inhibited PDGF-mediated PASMC proliferation. These results suggest that PDGF-mediated PASMC proliferation is associated with c-Jun/STAT3-induced upregulation of TRPC6 expression. The resultant increase in CCE raises [Ca2+]cyt, facilitates return of Ca2+ to the SR, and enhances PASMC growth.
Regulation of intracellular calcium in N1E-115 neuroblastoma cells: the role of Na+/Ca2+exchange
Authors: Kopper Kara L. , Adorante Joseph S.
Published: May 2013

In fura 2-loaded N1E-115 cells, regulation of intracellular Ca2+ concentration ([Ca2+]i) following a Ca2+ load induced by 1 μM thapsigargin and 10 μM carbonylcyanide p-trifluoromethyoxyphenylhydrazone (FCCP) was Na+ dependent and inhibited by 5 mM Ni2+. In cells with normal intracellular Na+ concentration ([Na+]i), removal of bath Na+, which should result in reversal of Na+/Ca2+exchange, did not increase [Ca2+]i unless cell Ca2+ buffer capacity was reduced. When N1E-115 cells were Na+ loaded using 100 μM veratridine and 4 μg/ml scorpion venom, the rate of the reverse mode of the Na+/Ca2+ exchanger was apparently enhanced, since an ∼4- to 6-fold increase in [Ca2+]ioccurred despite normal cell Ca2+ buffering. In SBFI-loaded cells, we were able to demonstrate forward operation of the Na+/Ca2+ exchanger (net efflux of Ca2+) by observing increases (∼ 6 mM) in [Na+]i. These Ni2+ (5 mM)-inhibited increases in [Na+]i could only be observed when a continuous ionomycin-induced influx of Ca2+ occurred. The voltage-sensitive dye bis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used to measure changes in membrane potential. Ionomycin (1 μM) depolarized N1E-115 cells (∼25 mV). This depolarization was Na+dependent and blocked by 5 mM Ni2+ and 250–500 μM benzamil. These data provide evidence for the presence of an electrogenic Na+/Ca2+ exchanger that is capable of regulating [Ca2+]i after release of Ca2+ from cell stores.
Metabolic inhibition with cyanide induces calcium release in pulmonary artery myocytes and Xenopus oocytes
Authors: Wang Yong-Xiao , Zheng Yun-Min , Abdullaev Iskandar , Kotlikoff Michael I.
Published: May 2013

We examined the effects of metabolic inhibition on intracellular Ca2+ release in single pulmonary arterial smooth muscle cells (PASMCs). Severe metabolic inhibition with cyanide (CN, 10 mM) increased intracellular calcium concentration ([Ca2+]i) and activated Ca2+-activated Cl− currents [ICl(Ca)] in PASMCs, responses that were greatly inhibited by BAPTA-AM or caffeine. Mild metabolic inhibition with CN (1 mM) increased spontaneous transient inward currents and Ca2+ sparks in PASMCs. In Xenopus oocytes, CN also induced Ca2+ release and activated ICl(Ca), and these responses were inhibited by thapsigargin and cyclopiazonic acid to deplete sarcoplasmic reticulum (SR) Ca2+, whereas neither heparin nor anti-inositol 1,4,5-trisphosphate receptor (IP3R) antibodies affected CN responses. In both PASMCs and oocytes, CN-evoked Ca2+release was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP) and oligomycin or CCCP and thapsigargin. Whereas hypoxic stimuli resulted in Ca2+release in pulmonary but not mesenteric artery myocytes, CN induced release in both cell types. We conclude that metabolic inhibition with CN increases [Ca2+]i in both pulmonary and systemic artery myocytes by stimulating Ca2+ release from the SR and mitochondria.
TRPC4 currents have properties similar to the pacemaker current in interstitial cells of Cajal
Authors: Walker Rebecca L. , Koh Sang Don , Sergeant Gerard P. , Sanders Kenton M. , Horowitz Burton
Published: May 2013

Interstitial cells of Cajal (ICC) are the pacemaker cells responsible for the generation and propagation of electrical slow waves in phasic muscles of the gastrointestinal (GI) tract. The pacemaker current that initiates each slow wave derives from a calcium-inhibited, voltage-independent, nonselective cation channel. This channel in ICC displays properties similar to that reported for the transient receptor potential (TRP) family of nonselective cation channels, particularly those seen for TRPC3 and TRPC4. We have identified transcripts for TRPC4 in individually isolated ICC and have cloned the two alternatively spliced forms of TRPC4, TRPC4α and TRPC4β, from GI muscles. TRPC4β is missing an 84-amino acid segment from the carboxy terminus. Expression of either form using the whole cell patch-clamp technique led to calcium-inhibited, nonselective cation channels as determined by N-methyl-d-glucamine replacement experiments and BAPTA dialysis. Expression of TRPC4β channels recorded at the whole cell level had characteristics similar to the nonselective cation current in ICC. The single-channel conductance of TRPC4β was determined to be 17.5 pS. Application of calmidazolium to cells expressing TRPC4β led to a significant increase in the inward current of these cells at both the whole cell and single-channel level, and currents were sensitive to block by 10 μM lanthanum, niflumic acid, and DIDS. Comparison of the properties reported for the nonselective cation current in ICC and those identified here for TRPC4β led us to conclude that a TRPC4-like current encodes the plasmalemmal pacemaker current in murine small intestine.
Role of VASP in reestablishment of epithelial tight junction assembly after Ca2+ switch
Authors: Lawrence Donald W. , Comerford Katrina M. , Colgan Sean P.
Published: May 2013

Epithelial permeability is tightly regulated by intracellular messengers. Critical to maintaining barrier integrity is the formation of tight junction complexes. A number of signaling pathways have been implicated in tight junction biogenesis; however, the precise molecular mechanisms are not fully understood. A growing body of evidence suggests a role for intracellular cAMP in tight junction assembly. Using an epithelial model, we investigated the role of cAMP signal transduction in barrier recovery after Ca2+ switch. Our data demonstrate that elevation of intracellular cAMP levels significantly enhanced barrier recovery after Ca2+ switch. Parallel experiments revealed that epithelial barrier recovery is diminished by H-89, a specific and potent inhibitor of cAMP-dependent protein kinase (protein kinase A) activity. Of the possible PKA effector proteins, the vasodilator-stimulated phosphoprotein (VASP) is an attractive candidate, since it has been implicated in actin-binding and cross-linking functions. We therefore hypothesized that VASP may play a role in the cAMP-mediated regulation of epithelial junctional reassembly after Ca2+ switch. We demonstrate here that VASP is phosphorylated via a PKA-dependent process under conditions that enhance barrier recovery. Confocal laser scanning microscopy studies revealed that VASP localizes with ZO-1 at the tight junction and at cell-cell borders and that phospho-VASP appears at the junction after Ca2+ switch. Subsequent transfection studies utilizing epithelial cells expressing truncated forms of VASP abnormal in oligomerization or actin-binding activity revealed a functional diminution of barrier recovery after Ca2+ chelation. Our present studies suggest that VASP may provide a link between cAMP signal transduction and epithelial permeability.
SH oxidation coordinates subunits of rat brain ryanodine receptor channels activated by calcium and ATP
Authors: Bull Ricardo , Marengo Juan José , Finkelstein José Pablo , Behrens María Isabel , Alvarez Osvaldo
Published: May 2013

We have reported that ryanodine receptor (RyR) channels display three different responses to cytoplasmic free Ca2+ concentration ([Ca2+]) depending on their redox state (Marengo JJ, Hidalgo C, and Bull R. Biophys J 74: 1263–1277, 1998), with low, moderate, and high maximal fractional open times ( Po). Activation by ATP of single RyR channels from rat brain cortex was tested in planar lipid bilayers with 10 or 0.1 μM cytoplasmic [Ca2+]. At 10 μM [Ca2+], low- Po channels presented lower apparent affinity to activation by ATP [[ATP] for half-maximal activation ( KaATP) = 422 μM] than moderate- Po channels ( KaATP = 82 μM). Oxidation of low- Po channels with thimerosal or 2,2′-dithiodipyridine (DTDP) gave rise to moderate- Po channels and decreased KaATP from 422 to 82 μM. At 0.1 μM cytoplasmic [Ca2+], ATP induced an almost negligible activation of low- Po channels. After oxidation to high- Po behavior, activation by ATP was markedly increased. Noise analysis of single-channel fluctuations of low- Po channels at 10 μM [Ca2+] plus ATP revealed the presence of subconductance states, suggesting a conduction mechanism that involves four independent subchannels. On oxidation the subchannels opened and closed in a concerted mode.
Age-dependent regulation of rat intestinal type IIb sodium-phosphate cotransporter by 1,25-(OH)2 vitamin D3
Authors: Xu Hua , Bai Liqun , Collins James F. , Ghishan Fayez K.
Published: May 2013

The current studies were designed to characterize type IIb sodium-inorganic phosphate (Pi) cotransporter (NaPi-IIb) expression and to assess the effect of 1,25-(OH)2 vitamin D3 on NaPi-IIb gene expression during rat ontogeny. Sodium-dependent Pi absorption by intestinal brush-border membrane vesicles (BBMVs) decreased with age, and NaPi-IIb gene expression also decreased proportionally with age. 1,25-(OH)2 vitamin D3 treatment increased intestinal BBMV Pi absorption by ∼2.5-fold in suckling rats and by ∼2.1-fold in adult rats. 1,25-(OH)2vitamin D3 treatment also increased NaPi-IIb mRNA abundance by ∼2-fold in 14-day-old rats but had no effect on mRNA expression in adults. Furthermore, in rat intestinal epithelial (RIE) cells, 1,25-(OH)2 vitamin D3 increased NaPi-IIb mRNA abundance, an effect that was abolished by actinomycin D. Additionally, human NaPi-IIb gene promoter activity in transiently transfected RIE cells showed ∼1.6-fold increase after 1,25-(OH)2 vitamin D3 treatment. In conclusion, we demonstrate that the age-related decrease in intestinal sodium-dependent Pi absorption correlates with decreased NaPi-IIb mRNA expression. Our data also suggest that the effect of 1,25-(OH)2 vitamin D3 on NaPi-IIb expression is at least partially mediated by gene transcription in suckling rats.
Tumor necrosis factor-α inhibits myogenesis through redox-dependent and -independent pathways
Authors: Langen Ramon C. J. , Schols Annemie M. W. J. , Kelders Marco C. J. M. , van der Velden Jos L. J. , Wouters Emiel F. M. , Janssen-Heininger Yvonne M. W.
Published: May 2013

Muscle wasting accompanies diseases that are associated with chronic elevated levels of circulating inflammatory cytokines and oxidative stress. We previously demonstrated that tumor necrosis factor-α (TNF-α) inhibits myogenic differentiation via the activation of nuclear factor-κB (NF-κB). The goal of the present study was to determine whether this process depends on the induction of oxidative stress. We demonstrate here that TNF-α causes a decrease in reduced glutathione (GSH) during myogenic differentiation of C2C12 cells, which coincides with an elevated generation of reactive oxygen species. Supplementation of cellular GSH with N-acetyl-l-cysteine (NAC) did not reverse the inhibitory effects of TNF-α on troponin I promoter activation and only partially restored creatine kinase activity in TNF-α-treated cells. In contrast, the administration of NAC before treatment with TNF-α almost completely restored the formation of multinucleated myotubes. NAC decreased TNF-α-induced activation of NF-κB only marginally, indicating that the redox-sensitive component of the inhibition of myogenic differentiation by TNF-α occurred independently, or downstream of NF-κB. Our observations suggest that the inhibitory effects of TNF-α on myogenesis can be uncoupled in a redox-sensitive component affecting myotube formation and a redox independent component affecting myogenic protein expression.
Calcium-independent inhibition of glucose transport in PC-12 and L6 cells by calcium channel antagonists
Authors: Ardizzone Timothy D. , Lu Xiao-Hong , Dwyer Donard S.
Published: May 2013

The goal of these studies was to determine whether different calcium channel antagonists affect glucose transport in a neuronal cell line. Rat pheochromocytoma (PC-12) cells were treated with L-, T-, and N-type calcium channel antagonists before measurement of accumulation of 2-[3H]deoxyglucose (2-[3H]DG). The L-type channel antagonists nimodipine, nifedipine, verapamil, and diltiazem all inhibited glucose transport in a dose-dependent manner (2–150 μM) with nimodipine being the most potent and diltiazem only moderately inhibiting transport. T- and N-type channel antagonists had no effect on transport. The L-type channel agonist l-BAY K 8644 also inhibited uptake of 2-[3H]DG. The ability of these drugs to inhibit glucose transport was significantly diminished by the presence of unlabeled 2-DG in the uptake medium. Some experiments were performed in the presence of EDTA (4 mM) or in uptake buffer without calcium. The absence of calcium in the uptake medium had no effect on inhibition of glucose transport by nimodipine or verapamil. To examine the effects of these drugs on a cell model of a peripheral tissue, we studied rat L6 muscle cells. The drugs inhibited glucose transport in L6 myoblasts in a dose-dependent manner that was independent of calcium in the uptake medium. These studies suggest that the calcium channel antagonists inhibit glucose transport in cells through mechanisms other than the antagonism of calcium channels, perhaps by acting directly on glucose transporters.
Two-photon molecular excitation imaging of Ca2+transients in Langendorff-perfused mouse hearts
Authors: Rubart Michael , Wang Exing , Dunn Kenneth W. , Field Loren J.
Published: May 2013

The ability to image calcium signals at subcellular levels within the intact depolarizing heart could provide valuable information toward a more integrated understanding of cardiac function. Accordingly, a system combining two-photon excitation with laser-scanning microscopy was developed to monitor electrically evoked [Ca2+]itransients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca2+]itransients were recorded at depths ≤100 μm from the epicardial surface with the fluorescent indicators rhod-2 or fura-2 in the presence of the excitation-contraction uncoupler cytochalasin D. Evoked [Ca2+]i transients were highly synchronized among neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50% ( t 90–50%) and from 50 to 10% ( t 50–10%) of the peak [Ca2+]i were (means ± SE) 73 ± 4 and 126 ± 10 ms, respectively, and at 2 Hz, 62 ± 3 and 94 ± 6 ms ( n = 19, P < 0.05 vs. 1 Hz) in rhod-2-loaded cardiomyocytes. [Ca2+]i decay was markedly slower in fura-2-loaded hearts ( t 90–50% at 1 Hz, 128 ± 9 ms and at 2 Hz, 88 ± 5 ms; t 50–10% at 1 Hz, 214 ± 18 ms and at 2 Hz, 163 ± 7 ms; n = 19, P < 0.05 vs. rhod-2). Fura-2-induced deceleration of [Ca2+]i decline resulted from increased cytosolic Ca2+ buffering, because the kinetics of rhod-2 decay resembled those obtained with fura-2 after incorporation of the Ca2+ chelator BAPTA. Propagating calcium waves and [Ca2+]i amplitude alternans were readily detected in paced hearts. This approach should be of general utility to monitor the consequences of genetic and/or functional heterogeneity in cellular calcium signaling within whole mouse hearts at tissue depths that have been inaccessible to single-photon imaging.
Increased susceptibility of cholangiocytes to tumor necrosis factor-α cytotoxicity after bile duct ligation
Authors: Alpini Gianfranco , Ueno Yoshiyuki , Tadlock Laura , Glaser Shannon S. , LeSage Gene , Francis Heather , Taffetani Silvia , Marzioni Marco , Alvaro Domenico , Patel Tushar
Published: May 2013

Tumor necrosis factor (TNF)-α plays a critical role in epithelial cell injury. However, the role of TNF-α in mediating cholangiocyte injury under physiological or pathophysiological conditions is unknown. Thus we assessed the effects of TNF-α alone or following sensitization by actinomycin D on cell apoptosis, proliferation, and basal and secretin-stimulated ductal secretion in cholangiocytes from normal or bile duct-ligated (BDL) rats. Cholangiocytes from normal or BDL rats were highly resistant to TNF-α alone. However, presensitization by actinomycin D increased apoptosis in cholangiocytes following BDL and was associated with an inhibition of proliferation and secretin-stimulated ductal secretion. Thus TNF-α mediates cholangiocyte injury and altered ductal secretion following bile duct ligation. These observations suggest that cholestasis may enhance susceptibility to cytokine-mediated cholangiocyte injury.
Involvement of human PECAM-1 in angiogenesis and in vitro endothelial cell migration
Authors: Cao Gaoyuan , O'Brien Christopher D. , Zhou Zhao , Sanders Samuel M. , Greenbaum Jordan N. , Makrigiannakis Antonis , DeLisser Horace M.
Published: May 2013

Platelet endothelial cell adhesion molecule (PECAM)-1 has been implicated in angiogenesis, but a number of issues remain unsettled, including the independent involvement of human PECAM-1 (huPECAM-1) in tumor angiogenesis and the mechanisms of its participation in vessel formation. We report for tumors grown in human skin transplanted on severe combined immunodeficiency mice that antibodies against huPECAM-1 (without simultaneous treatment with anti-VE-cadherin antibody) decreased the density of human, but not murine, vessels associated with the tumors. Anti-huPECAM-1 antibody also inhibited tube formation by human umbilical vein endothelial cells (HUVEC) and the migration of HUVEC through Matrigel-coated filters or during the repair of wounded cell monolayers. The involvement of huPECAM-1 in these processes was confirmed by the finding that expression of huPECAM-1 in cellular transfectants induced tube formation and enhanced cell motility. These data provide evidence of a role for PECAM-1 in human tumor angiogenesis (independent of VE-cadherin) and suggest that during angiogenesis PECAM-1 participates in adhesive and/or signaling phenomena required for the motility of endothelial cells and/or their subsequent organization into vascular tubes.
Depletion of cartilage collagen fibrils in mice carrying a dominant negative Col2a1 transgene affects chondrocyte differentiation
Authors: Barbieri Ottavia , Astigiano Simonetta , Morini Monica , Tavella Sara , Schito Anna , Corsi Alessandro , Martino Davide Di , Bianco Paolo , Cancedda Ranieri , Garofalo Silvio
Published: May 2013

We have generated transgenic mice harboring the deletion of exon 48 in the mouse α1(II) procollagen gene ( Col2a1). This was the first dominant negative mutation identified in the human α1(II) procollagen gene ( COL2A1). Patients carrying a single allele with this mutation suffer from a severe skeletal disorder called spondyloepiphyseal dysplasia congenita (SED). Transgenic mice phenotype was neonatally lethal with severe respiratory failure, short bones, and cleft palate. Transgene mRNA was expressed at high levels. Growth plate cartilage of transgenic mice presented morphological abnormalities and reduced number of collagen type II fibrils. Chondrocytes carrying the mutation showed altered expression of several differentiation markers, like fibroblast growth factor receptor 3 (Fgfr3), Indian hedgehog (Ihh), runx2, cyclin-dependent kinase inhibitor P21CIP/WAF (Cdkn1a), and collagen type X (Col10a1), suggesting that a defective extracellular matrix (ECM) depleted of collagen fibrils affects chondrocytes differentiation and that this defect participates in the reduced endochondral bone growth observed in chondrodysplasias caused by mutations in COL2A1.
Extracellular ATP signaling and P2X nucleotide receptors in monolayers of primary human vascular endothelial cells
Authors: Schwiebert Lisa M. , Rice William C. , Kudlow Brian A. , Taylor Amanda L. , Schwiebert Erik M.
Published: May 2013

ATP and its metabolites regulate vascular tone; however, the sources of the ATP released in vascular beds are ill defined. As such, we tested the hypothesis that all limbs of an extracellular purinergic signaling system are present in vascular endothelial cells: ATP release, ATP receptors, and ATP receptor-triggered signal transduction. Primary cultures of human endothelial cells derived from multiple blood vessels were grown as monolayers and studied using a bioluminescence detection assay for ATP released into the medium. ATP is released constitutively and exclusively across the apical membrane under basal conditions. Hypotonic challenge or the calcium agonists ionomycin and thapsigargin stimulate ATP release in a reversible and regulated manner. To assess expression of P2X purinergic receptor channel subtypes (P2XRs), we performed degenerate RT-PCR, sequencing of the degenerate P2XR product, and immunoblotting with P2XR subtype-specific antibodies. Results revealed that P2X4 and P2X5 are expressed abundantly by endothelial cell primary cultures derived from multiple blood vessels. Together, these results suggest that components of an autocrine purinergic signaling loop exist in the endothelial cell microvasculature that may allow for “self-regulation” of endothelial cell function and modulation of vascular tone.
Cardiac hypertrophy associated with impaired regulation of cardiac ryanodine receptor by calmodulin and S100A1
Authors: Yamaguchi Naohiro , Chakraborty Asima , Huang Tai-Qin , Xu Le , Gomez Angela C. , Pasek Daniel A. , Meissner Gerhard
Published: May 2013

The cardiac ryanodine receptor (RyR2) is inhibited by calmodulin (CaM) and S100A1. Simultaneous substitution of three amino acid residues (W3587A, L3591D, F3603A; RyR2ADA) in the CaM binding domain of RyR2 results in loss of CaM inhibition at submicromolar (diastolic) and micromolar (systolic) Ca2+, cardiac hypertrophy, and heart failure in Ryr2ADA/ADAmice. To address whether cardiac hypertrophy results from the elimination of CaM and S100A1 inhibition at diastolic or systolic Ca2+, a mutant mouse was generated with a single RyR2 amino acid substitution (L3591D; RyR2D). Here we report that in single-channel measurements RyR2-L3591D isolated from Ryr2D/Dhearts lost CaM inhibition at diastolic Ca2+only, whereas S100A1 regulation was eliminated at both diastolic and systolic Ca2+. In contrast to the ∼2-wk life span of Ryr2ADA/ADAmice, Ryr2D/Dmice lived longer than 1 yr. Six-month-old Ryr2D/Dmice showed a 9% increase in heart weight-to-body weight ratio, modest changes in cardiac morphology, and a twofold increase in atrial natriuretic peptide mRNA levels compared with wild type. After 4-wk pressure overload with transverse aortic constriction, heart weight-to-body weight ratio and atrial natriuretic peptide mRNA levels increased and echocardiography showed changes in heart morphology of Ryr2D/Dmice compared with sham-operated mice. Collectively, the findings indicate that the single RyR2-L3591D mutation, which distinguishes the effects of diastolic and systolic Ca2+, alters heart size and cardiac function to a lesser extent in Ryr2D/Dmice than the triple mutation in Ryr2ADA/ADAmice. They further suggest that CaM inhibition of RyR2 at systolic Ca2+is important for maintaining normal cardiac function.
Myofiber prestretch magnitude determines regional systolic function during ectopic activation in the tachycardia-induced failing canine heart
Authors: Howard Elliot J. , Kerckhoffs Roy C. P. , Vincent Kevin P. , Krishnamurthy Adarsh , Villongco Christopher T. , Mulligan Lawrence J. , McCulloch Andrew D. , Omens Jeffrey H.
Published: May 2013

Electrical dyssynchrony leads to prestretch in late-activated regions and alters the sequence of mechanical contraction, although prestretch and its mechanisms are not well defined in the failing heart. We hypothesized that in heart failure, fiber prestretch magnitude increases with the amount of early-activated tissue and results in increased end-systolic strains, possibly due to length-dependent muscle properties. In five failing dog hearts with scars, three-dimensional strains were measured at the anterolateral left ventricle (LV). Prestretch magnitude was varied via ventricular pacing at increasing distances from the measurement site and was found to increase with activation time at various wall depths. At the subepicardium, prestretch magnitude positively correlated with the amount of early-activated tissue. At the subendocardium, local end-systolic strains (fiber shortening, radial wall thickening) increased proportionally to prestretch magnitude, resulting in greater mean strain values in late-activated compared with early-activated tissue. Increased fiber strains at end systole were accompanied by increases in preejection fiber strain, shortening duration, and the onset of fiber relengthening, which were all positively correlated with local activation time. In a dog-specific computational failing heart model, removal of length and velocity dependence on active fiber stress generation, both separately and together, alter the correlations between local electrical activation time and timing of fiber strains but do not primarily account for these relationships.
Impaired contractile function due to decreased cardiac myosin binding protein C content in the sarcomere
Authors: Cheng Y. , Wan X. , McElfresh T. A. , Chen X. , Gresham K. S. , Rosenbaum D. S. , Chandler M. P. , Stelzer J. E.
Published: May 2013

Mutations in cardiac myosin binding protein C (MyBP-C) are a common cause of familial hypertrophic cardiomyopathy (FHC). The majority of MyBP-C mutations are expected to reduce MyBP-C expression; however, the consequences of MyBP-C deficiency on the regulation of myofilament function, Ca2+ homeostasis, and in vivo cardiac function are unknown. To elucidate the effects of decreased MyBP-C expression on cardiac function, we employed MyBP-C heterozygous null (MyBP-C+/−) mice presenting decreases in MyBP-C expression (32%) similar to those of FHC patients carrying MyBP-C mutations. The levels of MyBP-C phosphorylation were reduced 53% in MyBP-C+/− hearts compared with wild-type hearts. Skinned myocardium isolated from MyBP-C+/− hearts displayed decreased cross-bridge stiffness at half-maximal Ca2+ activations, increased steady-state force generation, and accelerated rates of cross-bridge recruitment at low Ca2+ activations (<15% and <25% of maximum, respectively). Protein kinase A treatment abolished basal differences in rates of cross-bridge recruitment between MyBP-C+/− and wild-type myocardium. Intact ventricular myocytes from MyBP-C+/− hearts displayed abnormal sarcomere shortening but unchanged Ca2+ transient kinetics. Despite a lack of left ventricular hypertrophy, MyBP-C+/− hearts exhibited elevated end-diastolic pressure and decreased peak rate of LV pressure rise, which was normalized following dobutamine infusion. Furthermore, electrocardiogram recordings in conscious MyBP-C+/− mice revealed prolonged QRS and QT intervals, which are known risk factors for cardiac arrhythmia. Collectively, our data show that reduced MyBP-C expression and phosphorylation in the sarcomere result in myofilament dysfunction, contributing to contractile dysfunction that precedes compensatory adaptations in Ca2+ handling, and chamber remodeling. Perturbations in mechanical and electrical activity in MyBP-C+/− mice could increase their susceptibility to cardiac dysfunction and arrhythmia.
Endogenous osteopontin promotes ozone-induced neutrophil recruitment to the lungs and airway hyperresponsiveness to methacholine
Authors: Barreno Ramon X. , Richards Jeremy B. , Schneider Daniel J. , Cromar Kevin R. , Nadas Arthur J. , Hernandez Christopher B. , Hallberg Lance M. , Price Roger E. , Hashmi Syed S. , Blackburn Michael R. , Haque Ikram U. , Johnston Richard A.
Published: May 2013

Inhalation of ozone (O3), a common environmental pollutant, causes pulmonary injury, pulmonary inflammation, and airway hyperresponsiveness (AHR) in healthy individuals and exacerbates many of these same sequelae in individuals with preexisting lung disease. However, the mechanisms underlying these phenomena are poorly understood. Consequently, we sought to determine the contribution of osteopontin (OPN), a hormone and a pleiotropic cytokine, to the development of O3-induced pulmonary injury, pulmonary inflammation, and AHR. To that end, we examined indices of these aforementioned sequelae in mice genetically deficient in OPN and in wild-type, C57BL/6 mice 24 h following the cessation of an acute (3 h) exposure to filtered room air (air) or O3 (2 parts/million). In wild-type mice, O3 exposure increased bronchoalveolar lavage fluid (BALF) OPN, whereas immunohistochemical analysis demonstrated that there were no differences in the number of OPN-positive alveolar macrophages between air- and O3-exposed wild-type mice. O3 exposure also increased BALF epithelial cells, protein, and neutrophils in wild-type and OPN-deficient mice compared with genotype-matched, air-exposed controls. However, following O3 exposure, BALF neutrophils were significantly reduced in OPN-deficient compared with wild-type mice. When airway responsiveness to inhaled acetyl-β-methylcholine chloride (methacholine) was assessed using the forced oscillation technique, O3 exposure caused hyperresponsiveness to methacholine in the airways and lung parenchyma of wild-type mice, but not OPN-deficient mice. These results demonstrate that OPN is increased in the air spaces following acute exposure to O3 and functionally contributes to the development of O3-induced pulmonary inflammation and airway and lung parenchymal hyperresponsiveness to methacholine.