Cardiac Na channel remodeling provides a critical substrate for generation of reentrant arrhythmias in border zones of the infarcted canine heart. Recent studies show that Nav1.5 assembly and function are linked to ankyrin-G, gap, and mechanical junction proteins. In this study our objective is to expound the status of the cardiac Na channel, its interacting protein ankyrinG and the mechanical and gap junction proteins at two different times post infarction when arrhythmias are known to occur; that is, 48 hr and 5 day post coronary occlusion. Previous studies have shown the origins of arrhythmic events come from the subendocardial Purkinje and epicardial border zone. Our Purkinje cell (Pcell) voltage clamp study shows that INa and its kinetic parameters do not differ between Pcells from the subendocardium of the 48hr infarcted heart (IZPCs) and control non-infarcted Pcells (NZPCs). Immunostaining studies revealed that disturbances of Nav1.5 protein location with ankyrin-G are modest in 48 hr IZPCs. Therefore, Na current remodeling does not contribute to the abnormal conduction in the subendocardial border zone 48 hr post myocardial infarction as previously defined. In addition, immunohistochemical data show that Cx40/Cx43 co-localize at the intercalated disc (IDs) of control NZPCs but separate in IZPCs. At the same time, Purkinje cell desmoplakin and desmoglein2 immunostaining become diffuse while plakophilin2 and plakoglobin increase in abundance at IDs. In the epicardial border zone 5 days post myocardial infarction, immunoblot and immunocytochemical analyses showed that ankyrin-G protein expression is increased and re-localized to submembrane cell regions at a time when Nav1.5 function is decreased. Thus, Nav1.5 and ankyrin-G remodeling occur later after myocardial infarction compared to that of gap and mechanical junctional proteins. Gap and mechanical junctional proteins remodel in IZPCs early, perhaps to help maintain Nav1.5 subcellular location position and preserve its function soon after myocardial infarction.
Female gender is a risk factor for long QT-related arrhythmias, but the underlying mechanisms remain uncertain. Here, we tested the hypothesis that gender-dependent function of the post-depolarization ‘late’ sodium current (INa-L) contributes.
Methods and results
Studies were conducted in mice in which the canonical cardiac sodium channel Scn5a locus was disrupted, and expression of human wild-type SCN5A cDNA substituted. Baseline QT intervals were similar in male and female mice, but exposure to the sodium channel opener anemone toxin ATX-II elicited polymorphic ventricular tachycardia in 0/9 males vs. 6/9 females. Ventricular INa-L and action potential durations were increased in myocytes isolated from female mice compared with those from males before and especially after treatment with ATX-II. Further, ATX-II elicited potentially arrhythmogenic early afterdepolarizations in myocytes from 0/5 male mice and 3/5 female mice.
These data identify variable late INa as a modulator of gender-dependent arrhythmia susceptibility.
Mouse; Late sodium current; Gender; Arrhythmias
To evaluate the ability of ClO2 to decontaminate pathogens known to cause healthcare-associated infections in a hospital room strains of Acinetobacter baumannii, Escherichia coli, Enterococcus faecalis, Mycobacterium smegmatis, and Staphylococcus aureus were spot placed in duplicate pairs at 10 sites throughout a hospital room and then exposed to ClO2 gas. Organisms were collected and evaluated for reduction in colony forming units following gas exposure. Six sterilization cycles with varied gas concentrations, exposure limits, and relative humidity levels were conducted. Reductions in viable organisms achieved ranged from 7 to 10-log reductions. Two sterilization cycles failed to produce complete inactivation of organisms placed in a bathroom with the door closed. Reductions of organisms in the bathroom ranged from 6-log to 10-log reductions. Gas leakage between hospital floors did not occur; however, some minor gas leakage from the door of hospital room was measured which was subsequently sealed to prevent further leakage. Novel technologies for disinfection of hospital rooms require validation and safety testing in clinical environments. Gaseous ClO2 is effective for sterilizing environmental contamination in a hospital room. Concentrations of ClO2 up to 385 ppm were safely maintained in a hospital room with enhanced environmental controls.
chlorine dioxide; sterilization; gas; nosocomial; hospital
Serious health conditions, such as clubfoot, could be a major source of uncertainty and stress for parents of children affected. How parents deal with uncertainty and stress as related to their child’s health condition is of interest for medical professionals and health communicators alike. While physicians remain a preferred source of health information, during medical encounters or via phone and email communication, many individuals seek out health information on the Internet, including in online support communities. This study explored the connections between Uncertainty Management Theory (UMT) constructs and the potential contribution of the sense of virtual community (SOVC) to the UMT framework. The results of this research suggest that the UMT needs to be adapted for use in online contexts. One way is to include theoretical constructs, such as the sense of virtual community, specifically developed to measure online interactions. A modified and updated Uncertainty Management Theory could be useful in exploring, analyzing and understanding online behaviors related to health conditions such as clubfoot and thus contribute substantially to what we know about caregivers in their role as uncertainty managers.
Although there is evidence that client expectations influence client outcomes, a valid and reliable scale for measuring the range of client expectations for both massage therapy and the behaviors of their massage therapists does not exist. Understanding how client expectations influence client outcomes would provide insight into how massage achieves its reported effects.
To develop and validate the Client Expectations of Massage Scale (CEMS), a measure of clients’ clinical, educational, interpersonal, and outcome expectations.
Offices of licensed massage therapists in Iowa.
A practice-based research methodology was used to collect data from two samples of massage therapy clients. For Sample 1, 21 volunteer massage therapists collected data from their clients before the massage. Factor analysis was conducted to test construct validity and coefficient alpha was used to assess reliability. Correlational analyses with the CEMS, previous measures of client expectations, and the Life Orientation Test–Revised were examined to test the convergent and discriminant validity of the CEMS. For Sample 2, 24 massage therapists distributed study materials for clients to complete before and after a massage therapy session. Structural equation modeling was used to assess the construct, discriminant, and predictive validity of the CEMS.
Sample 1 involved 320 and Sample 2 involved 321 adult massage clients.
Standard care provided by licensed massage therapists.
Numeric Rating Scale for pain and Positive and Negative Affect Schedule–Revised (including the Serenity subscale).
The CEMS demonstrated good construct, convergent, discriminant and predictive validity, and adequate reliability. Client expectations were generally positive toward massage and their massage therapists. Positive outcome expectations had a positive effect on clients’ changes in pain and serenity. High interpersonal expectations had a negative effect on clients’ changes in serenity.
Client expectations contribute to the nonspecific effects of massage therapy.
massage therapy; validity; practice-based research; pain; affect
The D1275N SCN5A mutation has been associated with a range of unusual phenotypes including conduction disease and dilated cardiomyopathy (DCM) as well as atrial and ventricular tachyarrhythmias. However, when D1275N is studied in heterologous expression systems, most studies show near-normal sodium channel function. Thus, the relationship of the variant to the clinical phenotypes remains uncertain.
Methods and results
We identified D1275N in a patient with atrial flutter, atrial standstill, conduction disease, and sinus node dysfunction. There was no major difference in biophysical properties between wild-type and D1275N channels expressed in CHO or tsA201 cells in the absence or presence of β1 subunits. To determine D1275N function in vivo, the Scn5a locus was modified to knock out the mouse gene, and the full-length wild-type (H) or D1275N (DN) human SCN5A cDNAs were then inserted at the modified locus using recombinase mediated cassette exchange. Mice carrying the DN allele displayed slow conduction, heart block, atrial fibrillation, ventricular tachycardia, and a DCM phenotype, with no significant fibrosis or myocyte disarray on histological examination. The DN allele conferred gene-dose dependent increases in SCN5A mRNA abundance, but reduced sodium channel protein abundance and peak sodium current amplitudes (H/H, −41.0±2.9 pA/pF at −30mV; DN/H, 19.2±3.1 pA/pF, P<0.001 versus H/H; DN/DN, −9.3±1.1 pA/pF, P<0.001 versus H/H).
Although D1275N produces near normal currents in multiple heterologous expression experiments, our data establish this variant as a pathological mutation that generates conduction slowing, arrhythmias, and a DCM phenotype by reducing cardiac sodium current.
genetics; ion channels; cardiomyopathy; electrophysiology
Although we and others have demonstrated that neural stem cells (NSCs) may impact such neurogenetic conditions as lysosomal storage diseases when transplanted at birth, it has remained unclear whether such interventions can impact well-established mid-stage disease, a situation often encountered clinically. Here we report that when NSCs were injected intracranially into the brain of adult symptomatic Sandhoff (Hexb−/−) mice, cells migrated far from the injection site and integrated into the host cytoarchitecture, restoring β-hexosaminidase enzyme activity and promoting neuropathologic and behavioral improvement. Mouse lifespan increased, neurological function improved, and disease progression was slowed. These clinical benefits correlated with neuropathological correction at the cellular and molecular levels, reflecting the multiple potential beneficial actions of stem cells, including enzyme cross-correction, cell replacement, tropic support, and direct anti-inflammatory action. Pathotropism, i.e., migration and homing of NSCs to pathological sites, could be imaged in real time by magnetic resonance imaging. Differentially expressed chemokines might play a role in directing the migration of transplanted stem cells to sites of pathology. Because many of the beneficial actions of NSCs observed in newborn brains were recapitulated in adult brains to the benefit of Sandhoff recipients, NSC-based interventions may also be useful in symptomatic subjects with established disease, even in symptomatic patients.
neural stem cell therapy; lysosomal storage disorders; Sandhoff disease; neurodegenerative disease; metabolic cross-correction
Although multiple lines of evidence suggest variable expression of the cardiac sodium channel gene SCN5A plays a role in susceptibility to arrhythmia, little is known about its transcriptional regulation.
We used in silico and in vitro experiments to identify possible non-coding sequences important for transcriptional regulation of SCN5A. The results were extended to mice in which a putative regulatory region was deleted.
Methods and Results
We identified 92 non-coding regions highly conserved (>70%) between human and mouse SCN5A orthologs. Three conserved non-coding sequences (CNS) showed significant (>5-fold) activity in luciferase assays. Further in vitro studies indicated one, CNS28 in intron 1, as potential regulatory region. Using Recombinase-Mediated Cassette Exchange (RMCE), we generated mice in which a 435 bp region encompassing CNS28 was removed. Animals homozygous for the deletion showed significant increases in SCN5A transcripts, NaV1.5 protein abundance, and sodium current measured in isolated ventricular myocytes. ECGs revealed a significantly shorter QRS (10.7±0.2ms in controls vs. 9.7±0.2ms in knockouts) indicating more rapid ventricular conduction. In vitro analysis of CNS28 identified a short 3′ segment within this region required for regulatory activity and including an E-box motif. Deletion of this segment reduced reporter activity to 3.6±0.3% of baseline in CHO cells and 16±3% in myocytes (both P<0.05), and mutation of individual sites in the E-box restored activity to 62±4% and 57±2% of baseline in CHO cells and myocytes, respectively (both P<0.05).
These findings establish that regulation of cardiac sodium channel expression modulates channel function in vivo, and identify a non-coding region underlying this regulation.
Gene Expression Regulation; Sodium Channels; Mice; Transgenic
The need for both valid and reliable measurement is crucial for the assessment of sun behavior. We used test-retest reliability of a self-administered survey to examine the reliability of reporting on behavior relevant to artificial ultraviolet tanning, sunburns and sun sensitivity among sorority and fraternity-affiliated university students. Subjects were members of sororities and fraternities who participated in an initial survey and skin examination. Students were prompted by specific recall queries. The students completed a second survey 2-4 weeks later. High reliability on test-retest for questions evaluating the number of artificial UV tanning sessions and the number of sunburns during specific time periods was found. Moderate reliability for measures reporting the use of self-tanning creams, can be largely explained by only one third of the students reporting they had ever used self-tanning creams. Overall this study suggests that members of sororities and fraternities report lifetime artificial ultraviolet tanning consistently when required to recall time period specific exposures prior to estimating their lifetime exposure.
reproducibility of results; skin neoplasms; ultraviolet rays
To examine the importance of tanning among students in relation to attitudes and knowledge regarding skin cancer prevention.
A cross-sectional survey.
College students at a major Midwestern university
Students were recruited to complete a self-administered questionnaire that included information on sun-sensitivity, knowledge and tanning attitudes and behaviors. Survey sampling statistical techniques that account for clustering among the 163 students recruited were used.
We found a high level of skin cancer prevention knowledge; however knowledge was not related to a reduction in the importance of tanning. In many cases, higher levels of knowledge corresponded to a greater emphasis on the importance of tanning. Sunscreen use was low among this population. Those who placed an importance on tanning more often checked that they believed that “sunless tanning creams are safer than the sun”.
This population’s belief that they look healthier and feel better with a tan strongly influences the desire to tan. Therefore, future cancer information campaigns or other prevention efforts should directly address the desire to tan by encouraging the use of sunless tanning products as an alternative method of tanning.
Knowledge; Attitudes; Cancer Prevention; Tanning; Ultraviolet Rays
Alcohol use disorders (AUDs) impact millions of individuals and there remain few effective treatment strategies. Despite evidence that neuronal nicotinic acetylcholine receptors (nAChRs) have a role in AUDs, it has not been established which subtypes of the nAChR are involved. Recent human genetic association studies have implicated the gene cluster CHRNA3–CHRNA5–CHRNB4 encoding the α3, α5, and β4 subunits of the nAChR in susceptibility to develop nicotine and alcohol dependence; however, their role in ethanol-mediated behaviors is unknown due to the lack of suitable and selective research tools. To determine the role of the α3, and β4 subunits of the nAChR in ethanol self-administration, we developed and characterized high-affinity partial agonists at α3β4 nAChRs, CP-601932, and PF-4575180. Both CP-601932 and PF-4575180 selectively decrease ethanol but not sucrose consumption and operant self-administration following long-term exposure. We show that the functional potencies of CP-601932 and PF-4575180 at α3β4 nAChRs correlate with their unbound rat brain concentrations, suggesting that the effects on ethanol self-administration are mediated via interaction with α3β4 nAChRs. Also varenicline, an approved smoking cessation aid previously shown to decrease ethanol consumption and seeking in rats and mice, reduces ethanol intake at unbound brain concentrations that allow functional interactions with α3β4 nAChRs. Furthermore, the selective α4β2* nAChR antagonist, DHβE, did not reduce ethanol intake. Together, these data provide further support for the human genetic association studies, implicating CHRNA3 and CHRNB4 genes in ethanol-mediated behaviors. CP-601932 has been shown to be safe in humans and may represent a potential novel treatment for AUDs.
α3β4* nicotinic acetylcholine receptor; ethanol; drug abuse; addiction; rat; alcohol & alcoholism; animal models; drug discovery/development; neuropharmacology; alpha3beta4* nicotinic acetylcholine receptor; ethanol; drug abuse; addiction; rat
Flying birds often form flocks, with social1, navigational2 and anti-predator3 implications. Further, flying in a flock can result in aerodynamic benefits, thus reducing power requirements4, as demonstrated by a reduction in heart rate and wingbeat frequency in pelicans flying in a V-formation5. But how general is an aerodynamic power reduction due to group-flight? V-formation flocks are limited to moderately steady flight in relatively large birds, and may represent a special case. What are the aerodynamic consequences of flying in the more usual ‘cluster’ 6,7 flock? Here, we use data from innovative back-mounted GPS and 6 degree of freedom inertial sensors to show that pigeons 1) maintain powered, banked turns like aircraft, imposing dorsal accelerations of up to 2g, effectively doubling body weight and quadrupling induced power requirements; 2) increase flap frequency with increases in all conventional aerodynamic power requirements; and 3) increase flap frequency when flying near, particularly behind, other birds. Therefore, unlike V-formation pelicans, pigeons do not gain an aerodynamic advantage from flying in a flock; indeed, the increased flap frequency – whether due to direct aerodynamic interactions or requirements for increased stability or control – suggests a considerable energetic cost to flight in a tight cluster flock.
Heparan sulfate can bind several adhesion molecules involved in lymphocyte trafficking. However, the in vivo function of endothelial heparan sulfate in lymphocyte homing and stimulation of the immune response has not been elucidated. Here, we generated mutant mice deficient in the enzyme Ext1, which is required for heparan sulfate synthesis, in a Tek-dependent and inducible manner. Chemokine presentation was diminished in the mutant mice, causing the lack of appropriate integrin-mediated adhesion, and resulted in a marked decrease in lymphocyte sticking to high endothelial venules and in recruitment of resident dendritic cells through lymphatic vessels to the lymph nodes. As a consequence, mutant mice displayed a severe impairment in lymphocyte homing and a compromised contact hypersensitivity response. By contrast, lymphocyte rolling was increased due to loss of electrostatic repulsion by heparan sulfate. These results demonstrate critical roles of endothelial heparan sulfate in immune surveillance and immune response generation.
Alcohol use disorders (AUDs) impact millions of individuals and there remain few effective treatment strategies. Despite evidence that neuronal nicotinic acetylcholine receptors (nAChRs) play a role in AUDs, it has not been established which subtypes of the nAChR are involved. Recent human genetic association studies have implicated the gene cluster CHRNA3-CHRNA5-CHRNB4 encoding the α3, α5 and β4 subunits of the nAChR in susceptibility to develop nicotine and alcohol dependence; however, their role in ethanol-mediated behaviors is unknown due to the lack of suitable and selective research tools. To determine the role of the α3, and β4 subunits of the nAChR in ethanol self-administration, we developed and characterized high affinity partial agonists at α3β4 nAChRs, CP-601932 and PF-4575180. Both CP-601932 and PF-4575180 selectively decrease ethanol but not sucrose consumption and operant self-administration following long-term exposure. We show that the functional potencies of CP-601932 and PF-4575180 at α3β4 nAChRs correlate with their unbound rat brain concentrations suggesting that the effects on ethanol self-administration are mediated via interaction with α3β4 nAChRs. Also varenicline, an approved smoking cessation aid previously shown to decrease ethanol consumption and seeking in rats and mice, reduces ethanol intake at unbound brain concentrations that allow functional interactions with α3β4 nAChRs. Furthermore, the selective α4β2* nAChR antagonist, DHβE, did not reduce ethanol intake. Together, these data provide further support for the human genetic association studies implicating CHRNA3 and CHRNB4 genes in ethanol-mediated behaviors. CP-601932 has been shown to be safe in humans and may represent a potential novel treatment for AUDs.
α3β4* nicotinic acetylcholine receptor; ethanol; drug abuse; addiction; rat
Social support has been identified as an important factor in facilitating recovery from injury. However, no previous authors have prospectively assessed the change in social support patterns before and after injury.
To examine the preinjury and postinjury social support patterns among male and female collegiate athletes.
Prospective observational study.
A Big Ten Conference university.
Patients or Other Participants:
A total of 256 National Collegiate Athletic Association Division I male and female collegiate athletes aged 18 or older from 13 sports teams.
Main Outcome Measure(s):
Injury incidence was identified using the Sports Injury Monitoring System. Social support was measured using the 6-item Social Support Questionnaire. Data on preinjury and postinjury social support patterns were compared.
Male athletes reported more sources of social support than female athletes, whereas female athletes had greater satisfaction with the support they received. Athletes' social support patterns changed after they became injured. Injured athletes reported relying more on coaches (P = .003), athletic trainers (P < .0001), and physicians (P = .003) for social support after they became injured. Athletes also reported greater postinjury satisfaction with social support received from friends (P = .019), coaches (P = .001), athletic trainers (P < .0001), and physicians (P = .003).
Our findings identify an urgent need to better define the psychosocial needs of injured athletes and also strongly suggest that athletic trainers have a critical role in meeting these needs.
psychology; athletic trainers
This article traces the development of a research project with a Native American community. Four principles were used to guide the development of the “Community Partnership to Affect Cherokee Adolescent Substance Abuse” project using a community-based participatory research approach. The principles suggest that establishing trust is key when developing and conducting research with a Native American community.
Very little is known about variation in prescription drug use and spending. We examine variation in outpatient prescription use and spending for diabetes and hyperlipidemia in the Veterans Affairs (VA) system and its association with quality measures for these conditions.
Study Design and Methods
We compared outpatient prescription use, spending and quality of care across 135 VA Medical Centers (VAMCs) in 2008, including 2.3 million patients dispensed lipid-lowering medications and 981,031 dispensed diabetes medications. We calculated VAMC-level cost/patient for these medications, proportion of patients on brand-name drugs, and Healthcare Effectiveness Data and Information Set (HEDIS) scores for hyperlipidemia (LDL<100mg/dl) and diabetes (HgA1c>9 or not measured) at each facility.
The median cost/patient for lipid-lowering agents in 2008 was $49.60 and varied from $39.68 in the least expensive quartile of VAMCs to $69.57 in the most expensive (p<0.001). For diabetes agents, median cost/patient was $158.34 and varied from $123.34 in the least expensive quartile to $198.31 in the most expensive (p<0.001). The proportion of patients dispensed brand name oral drugs in these classes in VAMCs in the most expensive quartile was twice that in the lowest quartile (p<0.001). There was no correlation between VAMC-level prescription spending and performance on HEDIS measures for lipid-lowering drugs (r=.12 and r=.07) or diabetes agents (r=-.10).
Despite a closely managed formulary, significant variation in prescription spending and use of brand name drugs exists in the VA. Although we could not explicitly risk adjust, there appears to be no association between spending on medications and quality of care.
Notch receptor signaling is required for T cell development, but its role in NK cell development is poorly understood. We compared the ability of the five mammalian Notch ligands (Jagged1, Jagged2, Delta1, Delta3, or Delta4) to induce NK cell development from human hematopoietic progenitor cells (HPCs). CD34+ HPCs were cultured with OP9 stromal cell lines transduced with one of the Notch ligands or with OP9 stromal cells alone, in the presence of IL-7, Flt3L, and IL-15. Differentiation and expansion of CD56+CD3− cells was greatly accelerated in the presence of Jagged2, Delta-1, or Delta-4, versus culture in the absence of ligand or in the presence of Jagged1 or Delta3. At four weeks, cultures containing Jagged2, Delta1, or Delta4 contained 80–90% NK cells, with the remaining cells being CD33+ myeloid cells. Notch-induced NK (N-NK) cells resembled CD56bright NK cells in that they were CD16-, CD94−, CD117+, and KIR-. They also expressed NKp30, NKp44, NKp46, 2B4, and DNAM-1, with partial expression of NKG2D. The N-NK cells displayed cytotoxic activity against the K562 and RPMI-8226 cell lines, at levels similar to activated peripheral blood NK cells, although killing of Daudi cells was not present. N-NK cells were also capable of IFN-γ secretion. Thus, Notch ligands have differential ability to induce and expand immature but functional NK cells from CD34+ HPCs. The use of Notch ligands to generate functional NK cells in vitro may be significant for cellular therapy purposes.
This study examined the relationship between Cherokee self-reliance and related values expressed through word-use in stories of stress written by Cherokee adolescents. The overall aim of this pilot study was to test the feasibility of using cultural appropriate measurements for a larger intervention study of substance abuse prevention in Cherokee adolescents. A sample of 50 Cherokee adolescent senior high school students completed the Cherokee Self-Reliance Questionnaire and wrote their story of stress. The Linguistic Inquiry and Word Count (LIWC) program, a word-based computerized text analysis software, was used to report the percentage of words used in the selected word categories in relation to all the words used by a participant. Word-use from the stories of stress were found to correlate with Cherokee self-reliance.
Cherokee Self-Reliance; Linguistic Inquiry and Word Count; Culture; Stress
Inositol 1,4,5-trisphosphate (InsP3) receptors are calcium-release channels found in the endoplasmic/sarcoplasmic reticulum (ER/SR) membrane of diverse cell types. InsP3 receptors release Ca2+ from ER/SR lumenal stores in response to InsP3 generated from various stimuli. The complex spatial and temporal patterns of InsP3 receptor-mediated Ca2+ release regulate many cellular processes, ranging from gene transcription to memory. Ankyrins are adaptor proteins implicated in the targeting of ion channels and transporters to specialized membrane domains. Multiple independent studies have documented in vitro and in vivo interactions between ankyrin polypeptides and the InsP3 receptor. Moreover, loss of ankyrin-B leads to loss of InsP3 receptor membrane expression and stability in cardiomyocytes. Despite extensive biochemical and functional data, the validity of in vivo ankyrin-InsP3 receptor interactions remains controversial. This controversy is based on inconsistencies between a previously identified ankyrin-binding region on the InsP3 receptor and InsP3 receptor topology data that demonstrate the inaccessibility of this lumenal binding site on the InsP3 receptor to cytosolic ankyrin polypeptides. Here we use two methods to revisit the requirements on InsP3 receptor for ankyrin binding. We demonstrate that ankyrin-B interacts with the cytoplasmic N-terminal domain of InsP3 receptor. In summary, our findings demonstrate that the ankyrin-binding site is located on the cytoplasmic face of the InsP3 receptor, thus validating the feasibility of in vivo ankyrin-InsP3 receptor interactions.
Trafficking; ankyrin; calcium; cytoskeleton; transport; InsP3 receptor
Leukocyte trafficking involves specific recognition between P-selectin and L-selectin and PSGL-1 containing core 2-based O-glycans expressing sialyl Lewis x (SLex) antigen. However, the structural identity of the glycan component(s) displayed by murine neutrophil PSGL-1 that contributes to its P-selectin counter-receptor activity has been uncertain, since these cells express little if any SLex antigen, and because there have been no direct studies to examine murine PSGL-1 glycosylation. To address this uncertainty, we studied PSGL-1 glycosylation in the murine cell line WEHI-3 using metabolic-radiolabeling with 3H-monosaccharide precursors to detect low-abundance O-glycan structures. We report that PSGL-1 from WEHI-3 cells expresses a di-sialylated core 2 O-glycan containing the SLex antigen. This fucosylated O-glycan is scarce on PSGL-1 and essentially undetectable in total leukocyte glycoproteins from WEHI-3 cells. These results demonstrate that WEHI-3 cells selectively fucosylate PSGL-1 to generate functionally important core 2-based O-glycans containing the SLex antigen.
Lewis x; murine; O-glycan; P-selectin; PSGL-1
The mechanisms involved in the pathogenesis of epilepsy, a chronic neurological disorder that affects approximately 1 percent of the world population, are not well understood1–3. Using a mouse model of epilepsy, we show that seizures induce elevated expression of vascular cell adhesion molecules and enhanced leukocyte rolling and arrest in brain vessels mediated by the leukocyte mucin P-selectin glycoprotein ligand-1 (PSGL-1) and leukocyte integrins α4β1 and αLβ2. Inhibition of leukocyte-vascular interactions either with blocking antibodies, or in mice genetically deficient in functional PSGL-1, dramatically reduced seizures. Treatment with blocking antibodies following acute seizures prevented the development of epilepsy. Neutrophil depletion also inhibited acute seizure induction and chronic spontaneous recurrent seizures. Blood-brain barrier (BBB) leakage, which is known to enhance neuronal excitability, was induced by acute seizure activity but was prevented by blockade of leukocyte-vascular adhesion, suggesting a pathogenetic link between leukocyte-vascular interactions, BBB damage and seizure generation. Consistent with potential leukocyte involvement in the human, leukocytes were more abundant in brains of epileptics than of controls. Our results suggest leukocyte-endothelial interaction as a potential target for the prevention and treatment of epilepsy.
Calcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over twenty years ago by activation dependence on Ca2+/CaM, but recent evidence shows CaMKII activity is also enhanced by pro-oxidant conditions. Here we show that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM. CaMKII is activated by angiotensin II (AngII) induced oxidation, leading to apoptosis in cardiomyocytes, both in vitro and in vivo. CaMKII oxidation is reversed by methionine sulfoxide reductase A (MsrA), and MsrA−/− mice show exaggerated CaMKII oxidation and myocardial apoptosis, impaired cardiac function, and increased mortality after myocardial infarction. Our data demonstrate a novel, dynamic mechanism for CaMKII activation by oxidation and highlight the critical importance of oxidation-dependent CaMKII activation to AngII and ischemic myocardial apoptosis.
Myocardial Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibition improves cardiac function following myocardial infarction (MI), but the CaMKII-dependent pathways that participate in myocardial stress responses are incompletely understood. To address this issue, we sought to determine the transcriptional consequences of myocardial CaMKII inhibition after MI. We performed gene expression profiling in mouse hearts with cardiomyocyte-delimited transgenic expression of either a CaMKII inhibitory peptide (AC3-I) or a scrambled control peptide (AC3-C) following MI. Of the 8,600 mRNAs examined, 156 were substantially modulated by MI, and nearly half of these showed markedly altered responses to MI with CaMKII inhibition. CaMKII inhibition substantially reduced the MI-triggered upregulation of a constellation of proinflammatory genes. We studied 1 of these proinflammatory genes, complement factor B (Cfb), in detail, because complement proteins secreted by cells other than cardiomyocytes can induce sarcolemmal injury during MI. CFB protein expression in cardiomyocytes was triggered by CaMKII activation of the NF-κB pathway during both MI and exposure to bacterial endotoxin. CaMKII inhibition suppressed NF-κB activity in vitro and in vivo and reduced Cfb expression and sarcolemmal injury. The Cfb–/– mice were partially protected from the adverse consequences of MI. Our findings demonstrate what we believe is a novel target for CaMKII in myocardial injury and suggest that CaMKII is broadly important for the genetic effects of MI in cardiomyocytes.